A multiclade env-gag VLP mRNA vaccine elicits tier-2 HIV-1-neutralizing antibodies and reduces the risk of heterologous SHIV infection in macaques

Abstract: The development of a protective vaccine remains a top priority for the control of the HIV/AIDS pandemic. Here, we show that a messenger RNA (mRNA) vaccine co-expressing membrane-anchored HIV-1 envelope (Env) and simian immunodeficiency virus (SIV) Gag proteins to generate virus-like particles (VLPs) induces antibodies capable of broad neutralization and reduces the risk of infection in rhesus macaques. In mice, immunization with co-formulated env and gag mRNAs was superior to env mRNA alone in inducing neutralizing antibodies. Macaques were primed with a transmitted-founder clade-B env mRNA lacking the N276 glycan, followed by multiple booster immunizations with glycan-repaired autologous and subsequently bivalent heterologous envs (clades A and C). This regimen was highly immunogenic and elicited neutralizing antibodies against the most prevalent (tier-2) HIV-1 strains accompanied by robust anti-Env CD4+ T cell responses. Vaccinated animals had a 79% per-exposure risk reduction upon repeated low-dose mucosal challenges with heterologous tier-2 simian-human immunodeficiency virus (SHIV AD8). Thus, the multiclade env-gag VLP mRNA platform represents a promising approach for the development of an HIV-1 vaccine.

Zhang P
Narayanan E
Liu Q
Tsybovsky Y
Boswell K
Ding S
Hu Z
Follmann D
Lin Y
Miao H
Schmeisser H
Rogers D
Falcone S
Elbashir SM
Presnyak V
Bahl K
Prabhakaran M
Chen X
Sarfo EK
Ambrozak DR
Gautam R
Martin MA
Swerczek J
Herbert R
Weiss D
Misamore J
Ciaramella G
Himansu S
Stewart-Jones G
McDermott A
Koup RA
Mascola JR
Finzi A
Carfi A
Fauci AS
Lusso P

Simian Acquired Immunodeficiency Syndrome

Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't


Fitting a vaccine into the HIV prevention landscape

Journal of the International AIDS Society

AIDS Vaccines
Antibodies, Neutralizing
HIV Antibodies
HIV Infections
HIV-1
Humans


From mRNA sensing to vaccines

Abstract: The 2005 Immunity paper by Karikó et al. has been hailed as a cornerstone insight that directly led to the design and delivery of the mRNA vaccines against COVID-19. We asked experts in pathogen sensing, vaccine development, and public health to provide their perspective on the study and its implications.

Fauci AS
Merad M
Swaminathan S
Hur S
Topol E
Fitzgerald K
Reis e Sousa C
Corbett KS
Bauer S

Animals
COVID-19
COVID-19 Vaccines
History, 21st Century
Humans
RNA, Messenger
SARS-CoV-2
Vaccine Development
World Health Organization
mRNA Vaccines

Historical Article
Journal Article
Portrait


Update on and Future Directions for Use of Anti-SARS-CoV-2 Antibodies: National Institutes of Health Summit on Treatment and Prevention of COVID-19

Abstract: As the fourth wave of the SARS-CoV-2 pandemic encircles the globe, there remains an urgent challenge to identify safe and effective treatment and prevention strategies that can be implemented in a range of health care and clinical settings. Substantial advances have been made in the use of anti-SARS-CoV-2 antibodies to mitigate the morbidity and mortality associated with COVID-19. On 15 June 2021, the National Institutes of Health, in collaboration with the U.S. Food and Drug Administration, convened a virtual summit to summarize existing knowledge on anti-SARS-CoV-2 antibodies and to identify key unanswered scientific questions to further catalyze the clinical development and implementation of antibodies.

Boggiano C
Eisinger RW
Lerner AM
Anderson JM
Woodcock J
Fauci AS
Collins FS

United States Food and Drug Administration


Distinct mechanisms of long-term virologic control in two HIV-infected individuals after treatment interruption of anti-retroviral therapy

Abstract: Certain infected individuals suppress human immunodeficiency virus (HIV) in the absence of anti-retroviral therapy (ART). Elucidating the underlying mechanism(s) is of high interest. Here we present two contrasting case reports of HIV-infected individuals who controlled plasma viremia for extended periods after undergoing analytical treatment interruption (ATI). In Participant 04, who experienced viral blips and initiated undisclosed self-administration of suboptimal ART detected shortly before day 1,250, phylogenetic analyses of plasma HIV env sequences suggested continuous viral evolution and/or reactivation of pre-existing viral reservoirs over time. Antiviral CD8+ T cell activities were higher in Participant 04 than in Participant 30. In contrast, Participant 30 exhibited potent plasma-IgG-mediated neutralization activity against autologous virus that became ineffective when he experienced sudden plasma viral rebound 1,434 d after ATI due to HIV superinfection. Our data provide insight into distinct mechanisms of post-treatment interruption control and highlight the importance of frequent monitoring of undisclosed use of ART and superinfection during the ATI phase.

Blazkova J
Gao F
Marichannegowda MH
Justement JS
Shi V
Whitehead EJ
Schneck RF
Huiting ED
Gittens K
Cottrell M
Benko E
Kovacs C
Lack J
Sneller MC
Moir S
Fauci AS
Chun TW

env Gene Products, Human Immunodeficiency Virus

Case Reports
Journal Article
Research Support, N.I.H., Intramural


Ending the Global HIV Epidemic Begins at the Individual National Level: An Update from the United States


Blocking α(4)β(7) integrin delays viral rebound in SHIV(SF162P3)-infected macaques treated with anti-HIV broadly neutralizing antibodies

Abstract: Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4β7 with an anti-α4β7 monoclonal antibody (Rh-α4β7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4β7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4β7 or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α4β7 and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α4β7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

Frank I
Cigoli M
Arif MS
Fahlberg MD
Maldonado S
Calenda G
Pegu A
Yang ES
Rawi R
Chuang GY
Geng H
Liu C
Zhou T
Kwong PD
Arthos J
Cicala C
Grasperge BF
Blanchard JL
Gettie A
Fennessey CM
Keele BF
Vaccari M
Hope TJ
Fauci AS
Mascola JR
Martinelli E

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't


Pre-vaccination and early B cell signatures predict antibody response to SARS-CoV-2 mRNA vaccine

Abstract: SARS-CoV-2 mRNA vaccines are highly effective, although weak antibody responses are seen in some individuals with correlates of immunity that remain poorly understood. Here we longitudinally dissected antibody, plasmablast, and memory B cell (MBC) responses to the two-dose Moderna mRNA vaccine in SARS-CoV-2-uninfected adults. Robust, coordinated IgA and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses, but earlier and more intensely after dose two. Distinct antigen-specific MBC populations also emerged post-vaccination with varying kinetics. We identified antigen non-specific pre-vaccination MBC and post-vaccination plasmablasts after dose one and their spike-specific counterparts early after dose two that correlated with subsequent antibody levels. These baseline and response signatures can thus provide early indicators of serological efficacy and explain response variability in the population.

medRxiv : the preprint server for health sciences

Kardava L
Rachmaninoff N
Lau WW
Buckner CM
Trihemasava K
de Assis FL
Wang W
Zhang X
Wang Y
Chiang CI
Narpala S
Reger R
McCormack GE
Seamon CA
Childs RW
Suffredini AF
Strich JR
Chertow DS
Davey RT
Sneller MC
O'Connell S
Li Y
McDermott A
Chun TW
Fauci AS
Tsang JS
Moir S


A Centenary Tale of Two Pandemics: The 1918 Influenza Pandemic and COVID-19, Part II

Abstract: Both the 1918 influenza pandemic and the 2019‒2021 COVID-19 pandemic are among the most disastrous infectious disease emergences of modern times. In addition to similarities in their clinical, pathological, and epidemiological features, the two pandemics, separated by more than a century, were each met with essentially the same, or very similar, public health responses, and elicited research efforts to control them with vaccines, therapeutics, and other medical approaches. Both pandemics had lasting, if at times invisible, psychosocial effects related to loss and hardship. In considering these two deadly pandemics, we ask: what lessons have we learned over the span of a century, and how are we applying those lessons to the challenges of COVID-19?

COVID-19
Communicable Disease Control
History, 20th Century
History, 21st Century
Humans
Influenza, Human
Pandemics
Public Health

Historical Article
Journal Article
Research Support, N.I.H., Intramural


Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit

Abstract: The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

Hall MD
Anderson JM
Anderson A
Baker D
Bradner J
Brimacombe KR
Campbell EA
Corbett KS
Carter K
Cherry S
Chiang L
Cihlar T
de Wit E
Denison M
Disney M
Fletcher CV
Ford-Scheimer SL
Götte M
Grossman AC
Hayden FG
Hazuda DJ
Lanteri CA
Marston H
Mesecar AD
Moore S
Nwankwo JO
O'Rear J
Painter G
Singh Saikatendu K
Schiffer CA
Sheahan TP
Shi PY
Smyth HD
Sofia MJ
Weetall M
Weller SK
Whitley R
Fauci AS
Austin CP
Collins FS
Conley AJ
Davis MI

Antiviral Agents
COVID-19
Drug Development
Humans
National Institutes of Health (U.S.)
Peptide Hydrolases
Protease Inhibitors
SARS-CoV-2
United States
Virus Replication
COVID-19 Drug Treatment


A Centenary Tale of Two Pandemics: The 1918 Influenza Pandemic and COVID-19, Part I

Abstract: Separated by a century, the influenza pandemic of 1918 and the COVID-19 pandemic of 2019-2021 are among the most disastrous infectious disease emergences of modern times. Although caused by unrelated viruses, the two pandemics are nevertheless similar in their clinical, pathological, and epidemiological features, and in the civic, public health, and medical responses to combat them. Comparing and contrasting the two pandemics, we consider what lessons we have learned over the span of a century and how we are applying those lessons to the challenges of COVID-19.

COVID-19
History, 20th Century
History, 21st Century
Humans
Influenza A virus
Influenza, Human
Pandemics
Public Health
SARS-CoV-2


COVID-19: The therapeutic landscape

Abstract: Therapeutics for hospitalized COVID-19 patients were identified through a robust research response with several lessons learned: clinical trial data should guide therapeutic use, results should not be extrapolated between disease stages, and robust studies should be designed to give clinically relevant data. These lessons should be applied to the outpatient research response.


The story behind COVID-19 vaccines

2019-nCoV Vaccine mRNA-1273
BNT162 Vaccine
Biomedical Research
COVID-19
COVID-19 Vaccines
Clinical Trials as Topic
Drug Development
Humans
Interdisciplinary Research
Vaccinology
Viral Vaccines


Human Immunodeficiency Virus/AIDS in the Era of Coronavirus Disease 2019: A Juxtaposition of 2 Pandemics

Abstract: The coronavirus disease 2019 (COVID-19) pandemic has significantly impacted persons with human immunodeficiency virus (HIV), interfering with critical health services for HIV prevention, treatment, and care. While there are multiple profiles of persons living with HIV and the impact of COVID-19 may differ for each, the severity of COVID-19 in persons with HIV is related strongly to the presence of comorbidities that increase the risk of severe disease in COVID-19 patients in the absence of HIV. An effective response to the juxtaposition of the HIV and COVID-19 pandemics requires a novel coordinated and collaborative global effort of scientists, industry, and community partners to accelerate basic and clinical research, as well as implementation science to operationalize evidence-based interventions expeditiously in real-world settings. Accelerated development and clinical evaluation of prevention and treatment countermeasures are urgently needed to mitigate the juxtaposition of the HIV and COVID-19 pandemics.

Acquired Immunodeficiency Syndrome
COVID-19
HIV
HIV Infections
Humans
Pandemics
SARS-CoV-2

Journal Article
Research Support, Non-U.S. Gov't


SARS-CoV-2 Variants of Concern in the United States-Challenges and Opportunities


SARS-CoV-2 Viral Variants-Tackling a Moving Target


Audio Interview: A Covid-19 Conversation with Anthony Fauci

Humans
Antiviral Agents
Attitude to Health
Carrier State
COVID-19
COVID-19 Testing
COVID-19 Vaccines
Disease Transmission, Infectious
Drug Development
Health Education
Immunization Programs
Public Health Practice
SARS-CoV-2
Sensitivity and Specificity
Treatment Outcome


SARS-CoV-2 Vaccines: Much Accomplished, Much to Learn

Abstract: As COVID vaccines roll out, internists and other health care providers are being turned to as trusted sources of information for patients and communities. Here, experts from NIAID outline the current state of knowledge regarding such vaccines. They contrast vaccine platforms, summarize clinical trial data regarding efficacy and safety, and comment on key questions including the ability of current vaccines to protect against infection and to decrease the prevalence of virus in the community.


The V2 loop of HIV gp120 delivers costimulatory signals to CD4(+) T cells through Integrin α(4)β(7) and promotes cellular activation and infection

Abstract: Acute HIV infection is characterized by rapid viral seeding of immunologic inductive sites in the gut followed by the severe depletion of gut CD4+ T cells. Trafficking of α4β7-expressing lymphocytes to the gut is mediated by MAdCAM, the natural ligand of α4β7 that is expressed on gut endothelial cells. MAdCAM signaling through α4β7 costimulates CD4+ T cells and promotes HIV replication. Similar to MAdCAM, the V2 domain of the gp120 HIV envelope protein binds to α4β7 In this study, we report that gp120 V2 shares with MAdCAM the capacity to signal through α4β7 resulting in CD4+ T cell activation and proliferation. As with MAdCAM-mediated costimulation, cellular activation induced by gp120 V2 is inhibited by anti-α4β7 monoclonal antibodies (mAbs). It is also inhibited by anti-V2 domain antibodies including nonneutralizing mAbs that recognize an epitope in V2 that has been linked to reduced risk of acquisition in the RV144 vaccine trial. The capacity of the V2 domain of gp120 to mediate signaling through α4β7 likely impacts early events in HIV infection. The capacity of nonneutralizing V2 antibodies to block this activity reveals a previously unrecognized mechanism whereby such antibodies might impact HIV transmission and pathogenesis.

Proceedings of the National Academy of Sciences of the United States of America

Goes LR
Sajani A
Sivro A
Olowojesiku R
Ray JC
Perrone I
Yolitz J
Girard A
Leyre L
Wibmer CK
Morris L
Gorini G
Franchini G
Mason RD
Roederer M
Mehandru S
Soares MA
Cicala C
Fauci AS
Arthos J

Anti-HIV Agents
Antibodies, Monoclonal
Antibodies, Neutralizing
CD4-Positive T-Lymphocytes
Cell Proliferation
Epitopes
HIV Envelope Protein gp120
HIV Infections
Host-Pathogen Interactions
Humans
Integrins
Lymphocyte Activation
Protein Domains
Signal Transduction
Simian Immunodeficiency Virus
Tretinoin

Journal Article
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't


Refocusing Human Microbiota Research in Infectious and Immune-mediated Diseases: Advancing to the Next Stage

Abstract: Changes in the microbiota are associated with disease susceptibility, immune system development, and responses to treatment. Refocusing research to elucidate the causal links between the human microbiota and infectious and immune-mediated diseases will be critical to harnessing its power to prevent, diagnose, and treat such diseases.

Giovanni MY
Schneider JS
Calder T
Fauci AS

Asthma
Communicable Diseases
Disease Susceptibility
Fecal Microbiota Transplantation
Humans
Hypersensitivity
Immune System
Immune System Diseases
Microbiota

Journal Article
Research Support, N.I.H., Extramural


Therapy for Early COVID-19: A Critical Need

Adaptive Clinical Trials as Topic
Antiviral Agents
COVID-19
Drug Discovery
Humans
Pandemics
Randomized Controlled Trials as Topic


Emerging Pandemic Diseases: How We Got to COVID-19


Preventing the Spread of SARS-CoV-2 With Masks and Other "Low-tech" Interventions

Aerosols
Betacoronavirus
COVID-19
Communicable Disease Control
Coronavirus Infections
Crowding
Hand Hygiene
Humans
Masks
Pandemics
Pneumonia, Viral
SARS-CoV-2
Social Isolation
Ventilation


Abstract: Infectious diseases prevalent in humans and animals are caused by pathogens that once emerged from other animal hosts. In addition to these established infections, new infectious diseases periodically emerge. In extreme cases they may cause pandemics such as COVID-19; in other cases, dead-end infections or smaller epidemics result. Established diseases may also re-emerge, for example by extending geographically or by becoming more transmissible or more pathogenic. Disease emergence reflects dynamic balances and imbalances, within complex globally distributed ecosystems comprising humans, animals, pathogens, and the environment. Understanding these variables is a necessary step in controlling future devastating disease emergences.

COVID-19
Communicable Diseases, Emerging
Coronavirus Infections
Demography
Environment
Host-Pathogen Interactions
Humans
Pandemics
Pneumonia, Viral


Research in the Context of a Pandemic

Dexamethasone
Humans
Pandemics
SARS-CoV-2
COVID-19 Drug Treatment


Structure and evolution-guided design of minimal RNA-guided nucleases

Abstract: The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.

Skopintsev P
Esain-Garcia I
DeTurk EC
Yoon PH
Zhou Z
Weiss T
Kamalu M
Chamraj A
Loi KJ
Langeberg CJ
Boger RS
Nisonoff H
Karp HM
Chen LX
Shi H
Vohra K
Banfield JF
Cate JHD
Jacobsen SE
Doudna JA

Humans
CRISPR-Cas Systems
Cryoelectron Microscopy
Directed Molecular Evolution
DNA
Gene Editing
Models, Molecular
Protein Conformation
Protein Engineering
RNA
RNA, Guide, CRISPR-Cas Systems
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins


Programming T cells for intercellular genome editing

Abstract: Therapeutic genome editing requires delivery of editing molecules to defined cell types, but targeting specificity and efficiency are currently limited. We hypothesized that properties inherent to immune cells, including tissue infiltration and programmed cell recognition, could be harnessed to engineer a cell-based delivery system. We show here that T cells can both produce and transfer editing machinery to target cells. In response to a programmable ligand, engineered T-lymphoid cells can transfer enzymes using complex spatiotemporal logic and deliver cargo in a cell contact-dependent or -independent manner. We demonstrate feasibility of this approach in primary human T cells, establishing a customizable genetic circuit for macromolecular delivery controlled by intercellular interactions.

bioRxiv : the preprint server for biology

Wasko KM
Maker M
Ngo W
Chen K
Ma E
Pattali R
Chen E
Leung T
Braverman J
Doudna JA


Harmonizing standards and resources for the medical genome

Abstract: Realizing the promise of precision medicine will require the highest standards of accuracy in genome sequencing and analysis. Here we describe challenges and opportunities for the field through the lens of genome data quality. We present recommendations in the context of specific areas of application for genomic sequencing in which isolated standards have arisen: germline sequencing, tumour sequencing, cell-free DNA testing, and sequencing for quality control in genetic therapy. Despite these distinct clinical contexts, technical challenges are often similar; for example, accurately detecting low-frequency genetic variants in tumour sequencing or gene-edited cells. We call for increased synchronization among these communities to establish new medical genome standards that promote confidence in genomic diagnostics and genetic therapies in a time of rapid technology-driven change. We suggest practical approaches for implementing these genome standards across contexts, and identify key areas that require further development.

Ashley EA
Alizadeh AA
Armitage H
Bhatt AS
Blumenfeld Y
Carroll A
Chavez RM
Giannikopoulos P
Grove ME
Halley MC
Khush K
Lennon NJ
Maragh S
Marson A
Paten B
Phillippy AM
Porteus MH
Rehm HL
Ringeisen BR
Salzman J
Schneider VA
Sedlazeck FJ
Steinmetz LM
Urnov FD
Wyman SK
Zook JM
Minor LB
Doudna JA

Humans
Genome, Human
Genomics
Neoplasms
Precision Medicine
Quality Control
Sequence Analysis, DNA


Publisher Correction: Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein

Chen K
Han H
Zhao S
Xu B
Yin B
Lawanprasert A
Trinidad M
Burgstone BW
Murthy N
Doudna JA


Targeting cancer-specific mutations with RNA-triggered chromatin shredding

Abstract: Genetic mutations that drive cancer often occur in tumour-suppressor proteins such as the p53 transcription factor, which is altered in 40-50% of cases1,2. However, current therapies often fail to target these mutations because the mutant proteins typically lack defined drug-binding pockets and restoring their endogenous function has proven challenging. Here we program Cas12a2, an RNA-guided CRISPR nuclease with trans-nucleolytic cleavage activity3,4, to kill cancer cells selectively by targeting cancer-specific transcripts. This approach limited cell growth by inducing trans shredding of chromatin and triggering DNA-damage responses and cell death. In contrast to existing methods, RNA-guided Cas12a2 senses cellular RNA signatures, enabling precise targeting of undruggable mutations. Transcript-activated chromatin shredding provides an innovative approach to precision disease treatments for undruggable targets.

Zeng J
Cheng Z
Chen H
Wang Z
Thompson J
Crosby KT
Han H
Singhal A
Ngo W
Xia C
Rosas-Rivera D
Zhang Z
Kang MH
Mao Y
Diolaiti ME
Lee GC
Diffley JFX
Song Y
Qiu L
Krah NM
Murthy N
Jackson RN
Liu Y
Ashworth A
Doudna JA


Selective Elimination of TP53 Mutant Cells by Transcript-Activated Chromatin Shredding

Abstract: Genetic mutations that drive cancer often occur in tumor suppressor proteins, including the p53 transcription factor which is altered in ~40-50% of cases1,2. However, current therapies fail to target most such mutations because the mutant proteins typically lack defined drug-binding pockets, and restoring the endogenous function has proven challenging. Here, we programmed CRISPR-Cas12a2, an RNA-guided nuclease with trans-nucleolytic cleavage activities3,4, to selectively kill cancer cells by targeting cancer-specific transcripts. This approach eliminates cells by inducing trans chromatin cleavage, triggering DNA damage and cell death. Unlike existing methods, RNA-guided Cas12a2 senses cellular RNA signatures to shred chromatin, enabling precise targeting of undruggable mutations. Transcript-activated chromatin shredding provides an innovative paradigm to develop precision disease treatments for undruggable targets.

Zeng J
Cheng Z
Chen H
Thompson J
Crosby KT
Han H
Ngo W
Xia C
Rosas-Rivera D
Kang MH
Mao Y
Lee G
Diffley JFX
Song Y
Qiu L
Krah NM
Murthy N
Jackson RN
Liu Y
Ashworth A
Doudna JA


A Noncontiguous Code for RNA-Guided DNA Recognition Preceded CRISPR

Abstract: CRISPR-Cas systems use RNA-guided proteins for adaptive immunity through a mechanism whose origin is unknown. Here we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein more ancient than CRISPR-Cas and vrRNAs comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that base pair with nucleic acid targets using an uninterrupted sequence, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NNs of each repeat collectively specify a target that itself contains a gapped recognition sequence. Analysis of natural vrRNA targets suggests VIPR acts against competing phages. We demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that the roots of adaptive immunity lie in ancient warfare between viruses, and reveal a new logic for programmable genetic control.

Yoon PH
Loi K
Zhang Z
Docter TA
Lopez SC
Langeberg CJ
Moezur-Rehman M
Vohra K
Zhou Z
Shi H
Boger R
Wang PY
Adler BA
Brohawn SG
Doudna JA


The phage nucleus synergizes with an anti-defense protein to resist bacterial immunity

Abstract: Chimallivirus bacteriophages enclose their replicating genomes in a protein-based compartment termed the phage nucleus. While the phage nucleus segregates phage DNA from host immune proteins, it is not known if additional factors are required to protect against DNA-targeting host defenses. Here, we identify a chimallivirus-encoded DarG2-like antitoxin that localizes to the phage nucleus and provides protection against phage-targeting DarTG2 toxin-antitoxin systems. This protein, which we term AdfM (anti-darT factor macro), contains a macrodomain and removes DarT2-mediated ADP-ribose modifications from DNA. In the absence of AdfM, DarT2 modifies phage DNA and restricts chimallivirus replication despite being largely excluded from the phage nucleus. Increasing the nuclear concentration of DarT2 while decreasing the nuclear concentration of AdfM reduces phage replication. These results show that the phage nucleus is insufficient to completely protect the chimallivirus genome from host defenses; rather, it is one component of a multilayered counter-defense strategy.

Morgan CJ
Rani P
Deep A
Liu R
Basu D
Chambers LR
Li YX
Levine M
Hsieh K
Adler BA
Birkholz E
Doudna JA
Villa E
Corbett KD
Pogliano J


Programmable kinetic barcoding for multiplexed RNA detection with Cas13a

Abstract: Rapid identification of viral infections and specific variants in patient samples requires a simple and multiplexed RNA detection method that does not rely on DNA sequencing. Although recent direct detection assays based on CRISPR-Cas13a offer rapid RNA detection by avoiding reverse transcription and DNA amplification required of gold-standard PCR assays, these assays are not easily multiplexed to detect multiple viruses or variants without dividing the sample into separate reactions. Here we show that Cas13a acting on single-target RNAs exhibits variable nuclease activity that depends on the interaction between the target RNA and crRNA. To exploit this feature for multiplexed detection, we devised a crRNA modification strategy that enables programmable tuning of Cas13a's nuclease enzymatic rates. Using a droplet-based Cas13a assay, we demonstrate that kinetic signatures can be harnessed to differentiate among respiratory viruses and SARS-CoV-2 variants in contrived and clinical samples. This kinetic barcoding strategy can be extended to additional RNA targets through simple modification of crRNAs.

Son S
Lyden A
Ng CF
Dextre A
Shu J
Stephens SI
Fozouni P
Knott GJ
Smock DCJ
Liu TY
Boehm D
Simoneau C
Kumar GR
Doudna JA
Ott M
Fletcher DA


Spligation enables programmable chimeric RNA generation in living cells

Abstract: The ability to precisely modify RNA offers opportunities to manipulate the flow of genetic information and influence transcript stability, localization and translation. RNA-targeting technologies enable RNA knockdown, base editing and trans-splicing, but more extensive transcript changes typically require genome editing or rely on the endogenous splicing machinery. Based on the ability of type III-A CRISPR-Csm complexes to catalyze programmable RNA cleavage in human cells, we investigated their potential to induce site-specific deletions while leaving the remainder of the transcript intact. Our data show that CRISPR-Csm complexes can generate short and long RNA excisions within a target transcript, and that the efficiency of this process is enhanced by fusion of Csm to the RNA ligase RtcB. Furthermore, cleavage of two different transcripts can trigger subsequent trans-ligation of the cleaved products into a chimeric transcript ("spligation"). Finally, we apply spligation to endogenous transcripts, using Csm to generate recombinant mRNA in cells independent of canonical splice sites. Collectively, this approach enables new forms of precise RNA manipulation in cells with potential applications in human disease.

Colognori DA
Wasko KM
Trinidad MI
Zhou Z
Doudna JA


In vivo site-specific engineering to reprogram T cells

Abstract: Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise1. However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR-Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.

Nyberg WA
Bernard PL
Ngo W
Wang CH
Ark J
Rothrock A
Borgo GM
Kimmerly GR
Jung JH
Allain V
Hamilton JR
Baldwin A
Stickels R
Wyman S
Khan SH
Lang S
Marsh D
Almudhfar N
Novick C
Mortazavi Y
Zhang S
AbdElwakil MM
Sandoval LR
Hwang S
Chu SN
Jung H
Liu C
Sharma D
McCreary T
Li Z
Satpathy AT
Carnevale J
Rutishauser RL
Cromer MK
Roybal KT
Dodgson SE
Doudna JA
Asokan A
Eyquem J

Animals
Mice
T-Lymphocytes
Receptors, Chimeric Antigen
CRISPR-Cas Systems
Humans
Transgenes
Gene Editing
Dependovirus
Female
Cellular Reprogramming
Genetic Vectors
Cell Engineering
Immunotherapy, Adoptive
Male


Efficient Transgene-Free Multiplexed Germline Editing via Viral Delivery of an Engineered TnpB

Weiss T
Kamalu M
Shi H
Wirnowski G
Ingelsson A
Amerasekera J
Vohra K
Trinidad MI
Li Z
Freitas E
Steinmetz N
Ambrose C
Chen K
Doudna JA
Jacobsen SE


Engineered TnpB genome editors for plants and human cells identified by ribonucleoprotein mutational scanning

Abstract: TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Because of their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold great potential for genome editing. However, most TnpBs lack robust gene-editing activity. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein using deep mutational scanning and we discovered activating mutations in both the RNA and the protein. Leveraging the protein's mutational landscape, we constructed a combinatorial library of activating mutations, from which we identified two enhanced TnpB variants. These variants increased editing in human cells, Nicotania benthamiana, pepper and rice. While editing efficiencies varied by target site, engineered variants achieved up to 55% insertion and deletion frequencies (a 50-fold increase over wild type) in N. benthamiana, surpassing ISYmu1 (<7%), AsCas12f-HKRA (<9%) and other compact editors. These findings highlight elements critical for regulating TnpB endonuclease activity and demonstrate latent activity accessible through mutation.

Thornton BW
Weissman RF
Rodriguez JE
Tran RV
Duong BT
Terrace CI
Nagalakshmi U
Austin G
Groover ED
Wang FZ
Park JU
Georgieva V
Tartaglia J
Cho MJ
Dinesh-Kumar SP
Doudna JA
Savage DF


Regulation of virion production by the ORF8 signal peptide across SARS-CoV-2 variants

Abstract: The open reading frame 8 (ORF8), an accessory protein of SARS-CoV-2, is prone to deletions and mutations across different viral variants, which was first described in several Singapore variants. The reason why viral evolution favors loss or inactivation of ORF8 is not fully understood, although the effects of ORF8 on inflammation, immune evasion, and disease severity have been described. Here we show -using clinical ORF8-deficient viral isolates, virus-like particles (VLPs) and viral replicons- that ORF8 expression dampens viral particle production. ORF8 physically interacts with the viral Spike protein and induces Golgi fragmentation, overall contributing to less virus particle production. Using systematic ORF8 deletions, we mapped the particle-reducing function to its N-terminal signal peptide. Interestingly, this part of ORF8 is severely truncated in the recent XBB.1.5 variant, and when restored, suppresses viral particle production in the context of the entire viral genome. Collectively, our data supports the model that evolutionary pressure exists to delete ORF8 sequence and expression across SARS-CoV-2 variants to fully enable viral particle production.

Khalid MM
Chen IP
Soveg FS
Taha TY
Tabata T
Suryawanshi RK
Syed AM
Ciling A
McCavitt-Malvido M
Schulze-Gahmen U
Hayashi J
Kim IJ
Fong SW
Batra J
Kumar GR
Renia L
Ng LF
Krogan NJ
Doudna JA
Verdin E
Ott M


Amplified genome editing by in vivo editor production

Abstract: Genome editing enzymes have vast therapeutic potential. However, achieving sufficient delivery in vivo remains a major challenge, because editing machinery is confined to the subset of transfectable cells in a tissue. Here, we tested the possibility that genome editing could be amplified in vivo by programming transfected cells to produce and transfer editing enzymes in lipid vesicles to neighboring cells. Our data show that this NANoparticle-Induced Transfer of Enzyme (NANITE) strategy tripled editing efficiency in cultured cells relative to non-spreading controls. Furthermore, a single intravenous injection of the NANITE plasmid into mice induced ~3-fold higher levels of liver editing at the Ttr locus relative to non-spreading controls, with corresponding reductions in serum transthyretin levels. Amplifying therapeutic enzymes in situ offers a nonviral and non-infectious strategy to overcome low delivery efficiencies and reduce effective dose requirements.

Ngo W
Rosas-Rivera D
Wasko KM
Qiu L
Kang MH
Gogna S
Zeng J
Hooks MT
Wu JLY
Li Z
Doudna JA


Efficient transgene-free multiplexed germline editing via viral delivery of an engineered TnpB

Abstract: Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing (Hu et al. 2025; Liu et al. 2025; Weiss et al. 2025). We recently established a VIGE approach for heritable editing at single loci in Arabidopsis by delivering the compact genome editor ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV) (Weiss et al. 2025). Here, we greatly improved this approach by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) (Zhou et al. 2026) to develop an efficient multiplexed genome editing platform.


Abstract: The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome editing capabilities. However, generating diverse multi-domain proteins with robust enzymatic properties remains challenging. Here we use an artificial intelligence-driven strategy that couples structure-guided inverse protein folding with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease. High-throughput functional screening of AI-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant and human cells. Cryo-EM-based structure determination of the most divergent active variant revealed new stabilizing contacts in the RNA/DNA interfaces across conformational states, demonstrating the design potential of this approach. Together these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space. An evolution- and structure-conditioned model enables design of active RNA-guided nucleases with new nucleic acid contacts resolved by cryo-EM.

Skopintsev P
Esain-Garcia I
DeTurk EC
Yoon PH
Zhou Z
Weiss T
Kamalu M
Chamraj A
Loi KJ
Langeberg CJ
Boger R
Nisonoff H
Karp HM
Chen L
Shi H
Vohra K
Banfield JF
Cate JHD
Jacobsen SE
Doudna JA


Stepwise DNA unwinding gates TnpB genome-editing activity

Abstract: TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through formation of a partially unwound intermediate state to a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in vivo. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

Zhou Z
Saffarian-Deemyad I
Shi H
Weiss T
Ur-Rehman MM
Vohra K
Skopintsev P
Yoon PH
Trinidad MI
Langeberg CJ
Kamalu M
Amerasekera J
Doherty EE
Aris KDP
Al-Sayyad N
Thornton BW
Weissman RF
Wasko KM
Esain-Garcia I
DeTurk EC
Savage DF
Jacobsen SE
Bryant Z
Doudna JA


Targeted delivery of genome editors in vivo

Abstract: Genome editing has revolutionized the treatment of genetic diseases, yet the difficulty of tissue-specific delivery currently limits applications of editing technology. In this Review, we discuss preclinical and clinical advances in delivering genome editors with both established and emerging delivery mechanisms. Targeted delivery promises to considerably expand the therapeutic applicability of genome editing, moving closer to the ideal of a precise 'magic bullet' that safely and effectively treats diverse genetic disorders.

Humans
Gene Editing
Animals
Genetic Therapy
Gene Transfer Techniques
CRISPR-Cas Systems


Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing

Abstract: CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST (VchCAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors and increased editing efficiency by 55.2-fold in E. coli, 5.6-fold in Pseudomonas putida, and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting VchCAST activity and enhances its efficiency as a bacterial genome editor.

Song LCT
Alker ATP
Oromí-Bosch A
Swartz SE
Martinson JNV
Arora J
Wang AM
Rovinsky R
Smith SJ
Pierce EC
Deutschbauer AM
Doudna JA
Cress BF
Rubin BE

Gene Editing
Transposases
Escherichia coli
Vibrio cholerae
CRISPR-Cas Systems
Genome, Bacterial
CRISPR-Associated Proteins


Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells

Abstract: Primary human myeloid cells are promising candidates for immunotherapy, yet efficient and scalable technologies for genetic engineering and screening in these cells are limited. Here we present a virus-like particle (VLP)-based toolkit that delivers diverse CRISPR genome editing modalities to human monocytes, macrophages, and dendritic cells with high efficiency while preserving viability and innate immune responsiveness. VLP-mediated delivery of ribonucleoprotein payloads supports gene knockout, base editing and epigenetic silencing, and enables site-specific integration of large DNA sequences when combined with AAV donors for homology-directed repair. Leveraging sgRNA delivery via VPX-lentivirus combined with Cas9 protein delivery via engineered virus-like particle (eVLP) treatment ("SLICeVLP"), we performed the first pooled loss-of-function screens in human macrophages. We uncovered regulators of TNF production and CD80 expression in human macrophages, converging on TNFAIP3 as a central regulator of inflammatory polarization. TNFAIP3 ablation promoted a pro-inflammatory cell state that is resistant to suppressive polarization, and augmented cytotoxicity of engineered HER2 CAR-macrophages. Taken together, this technology platform enables unbiased discovery and characterization of functional gene targets in primary human myeloid cells.

Jung H
Devant P
Ching C
Ota M
Hamilton J
Steinhart Z
Ngo W
Sandoval L
Jung JH
Xu D
An M
Urs E
Chen PA
Allain V
Tada T
Nuñez JK
Landau NR
Liu DR
Eyquem J
Doudna JA
Marson A
Carnevale J


Divergent viral phosphodiesterases for immune signaling evasion

Abstract: Cyclic dinucleotides (CDNs) and other short oligonucleotides play fundamental roles in immune system activation in organisms ranging from bacteria to humans. In response, viruses use phosphodiesterase (PDE)-mediated oligonucleotide cleavage for immune evasion, a strategy whose diversity has not yet been explored. Here, we use a canonical 2H PDE (2H PDE) structure-based search of prokaryotic and eukaryotic viral sequences to identify an exceptional diversity of 2H PDEs across the virome, including enzymes not detectable with sequence search methods alone. Despite active site conservation, biochemical experiments reveal remarkable substrate specificity of these PDEs that corresponds to variations in the core 2H fold. This nuanced specificity allows 2H PDEs to selectively degrade oligonucleotide messengers to avoid interfering with host nucleotide signaling. Together, these findings nominate viral 2H PDEs as key regulators of CDN signaling across the tree of life.

Doherty EE
Nomburg J
Adler BA
Lopez S
Hsieh K
Price N
Blount N
Doudna JA

Immune Evasion
Phosphoric Diester Hydrolases
Signal Transduction
Humans
Substrate Specificity
Viruses
Catalytic Domain
Viral Proteins


Temporal photoproximity labeling of ligand-activated EGFR neighborhoods using MultiMap

Abstract: Photoproximity labeling proteomics (PLP) methods have recently shown that cell surface receptors can form lateral interactome networks. Here, we present a paired set of PLP workflows that dynamically track neighborhood changes for oncogenic epidermal growth factor receptor (EGFR) over time, both outside and inside of cells. We achieved this by augmenting the multiscale PLP workflow we call MultiMap, where three photoprobes with different labeling ranges were photoactivated by one photocatalyst, eosin Y, anchored extracellularly and intracellularly on EGFR. We identified hundreds of neighboring proteins that changed within minutes to over 1 h after the addition of EGF. These neighborhoods reveal dynamic interactomes during early, middle and late signaling that drive phosphorylation, internalization, degradation and transcriptional regulation. This rapid 'molecular photographic' labeling approach provides snapshots of signaling neighborhoods, revealing their dynamic nature and potential for drug targeting.

Lin Z
Ngo W
Chou YT
Wu H
Susa KJ
Jun YW
Bivona TG
Doudna JA
Wells JA

ErbB Receptors
Humans
Ligands
Proteomics
Signal Transduction
Epidermal Growth Factor
Phosphorylation
Staining and Labeling


Characterizing and controlling CRISPR repair outcomes in nondividing human cells

Abstract: Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.

Ramadoss GN
Namaganda SJ
Kumar MM
Hamilton JR
Sharma R
Chow KG
Workley LA
Macklin BL
Sun M
Ha AS
Liu JC
Fellmann C
Watry HL
Dierks PH
Bose RS
Jin J
Perez BS
Sandoval Espinoza CR
Matia MP
Lu SH
Judge LM
Shy BR
Nussenzweig A
Adamson B
Murthy N
Doudna JA
Kampmann M
Conklin BR

Humans
Induced Pluripotent Stem Cells
Gene Editing
DNA Repair
CRISPR-Cas Systems
Neurons
Myocytes, Cardiac
DNA Damage
T-Lymphocytes


Recurrent acquisition of nuclease-protease pairs in antiviral immunity

Abstract: Antiviral immune systems diversify by integrating new genes into existing pathways, creating new mechanisms of viral resistance. We identified genes encoding a predicted nuclease paired with a trypsin-like protease repeatedly acquired by multiple, otherwise unrelated antiviral immune systems in bacteria. Cell-based and biochemical assays revealed that the nuclease is a proenzyme that cleaves DNA only after activation by its partner protease. Two distinct immune systems, Hachiman and AVAST (antiviral adenosine triphosphatase/nucleoside triphosphatase of the STAND superfamily, Avs), use the same mechanism of proteolytic activation despite their independent evolutionary origins. Examination of nuclease-protease inheritance patterns identified caspase-nuclease (canu) genomic loci that confer antiviral defense in a pathway reminiscent of eukaryotic caspase activation. These results uncover the coordinated activities of pronucleases and their activating proteases within different immune systems and show how coevolution enables defense system innovation.

Tuck OT
Hu JJ
Lopez SC
Adler BA
O'Brien CE
Hsieh K
Meredith C
Loi KJ
Yoon PH
Doherty EE
Lahiri A
Doudna JA

Bacteria
Bacterial Proteins
Caspases
Deoxyribonucleases
DNA Cleavage
Evolution, Molecular
Peptide Hydrolases
Proteolysis
Bacteriophages


A miniature CRISPR-Cas10 enzyme confers immunity by inhibitory signalling

Abstract: Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signalling that share mechanistic features with human antiviral systems1. In these pathways, including cyclic oligonucleotide-based antiphage signalling systems (CBASSs) and type III CRISPR systems in bacteria and cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response2-4. Here, in an unexpected inversion of this process, we show that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that represses a toxic effector. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing the spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TMβ. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TMβ-mediated cell death due to inner membrane collapse. These findings unveil a powerful defence strategy against virus-mediated immune suppression, expanding our understanding of the role of oligonucleotides in immunity.

Doherty EE
Adler BA
Yoon PH
Hsieh K
Loi K
Armbruster EG
Lahiri A
Bolling CS
Wilcox XE
Akkati A
Iavarone AT
Pogliano J
Doudna JA

Humans
Adenylyl Cyclases
Cell Death
CRISPR-Cas Systems
Dinucleoside Phosphates
HEK293 Cells
Immune Evasion
Membrane Proteins
Models, Molecular
Oligonucleotides
Signal Transduction
STING Protein

Research Support, U.S. Gov't, Non-P.H.S.


Abstract: Cyclic dinucleotides (CDNs) and other short oligonucleotides play fundamental roles in immune system activation in organisms ranging from bacteria to humans. In response, viruses use phosphodiesterase-mediated oligonucleotide cleavage for immune evasion, a strategy whose diversity has not yet been explored. We used a canonical 2H phosphodiesterase (2H PDE) structure-based search of prokaryotic and eukaryotic viral sequences to identify an exceptional diversity of 2H PDEs across the virome, including enzymes not detectable with sequence search methods alone. Despite active site conservation, biochemical experiments revealed remarkable substrate specificity of these PDEs that corresponds to variation in the core 2H fold. This nuanced specificity allows 2H PDEs to selectively degrade oligonucleotide messengers to avoid interfering with host immune signaling. Together, these findings nominate viral 2H PDEs as key regulators of CDN signaling across the tree of life.


Abstract: Antiviral immune systems diversify by integrating new genes into existing pathways, creating new mechanisms of viral resistance. We identified genes encoding a predicted nuclease paired with a trypsin-like protease repeatedly acquired by multiple, otherwise unrelated antiviral immune systems in bacteria. Cell-based and biochemical assays revealed the nuclease is a proenzyme that cleaves DNA only after activation by its partner protease. Phylogenetic analysis showed that two distinct immune systems, Hachiman and AVAST, use the same mechanism of proteolytic activation despite their independent evolutionary origins. Examination of nuclease-protease inheritance patterns identified caspase-nuclease (canu) genomic loci that confer antiviral defense in a pathway reminiscent of eukaryotic caspase activation. These results uncover the coordinated activities of pronucleases and their activating proteases within different immune systems and show how coevolution enables defense system innovation.

Tuck OT
Hu JJ
Adler BA
O'Brien CE
Lopez SC
Hsieh K
Meredith C
Lahiri A
Doherty EE
Doudna JA


Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing

Abstract: Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infect Escherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targeted E. coli cells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.

Roberts A
Adler BA
Cress BF
Doudna JA
Barrangou R

Gene Editing
Transposases
CRISPR-Cas Systems
Escherichia coli
Bacteriophage lambda
Genome, Bacterial


Directed evolution expands CRISPR-Cas12a genome-editing capacity

Abstract: CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

Ma E
Chen K
Shi H
Wasko KM
Esain-Garcia I
Trinidad MI
Zhou K
Ye J
Doudna JA

CRISPR-Cas Systems
Gene Editing
Humans
CRISPR-Associated Proteins
Directed Molecular Evolution
Endodeoxyribonucleases
Bacterial Proteins
Genome, Human


Improving RNA Secondary Structure Prediction Through Expanded Training Data

Abstract: In recent years, deep learning has revolutionized protein structure prediction, achieving remarkable speed and accuracy. RNA structure prediction, however, has lagged behind. Although several methods have shown some success in predicting RNA secondary and tertiary structures, none have reached the accuracy observed with contemporary protein models. The lack of success of these RNA structure prediction models has been proposed to be due to limited high-quality structural information that can be used as training data. To probe this proposed limitation, we developed a large and diverse dataset comprising paired RNA sequences and their corresponding secondary structures. We assess the utility of this enhanced dataset by retraining on a deep learning model, SincFold. We find that SincFold exhibited improved generalization to some previously unseen RNA families, enhancing its capability to predict accurate de novo RNA secondary structures. The RNASSTR dataset provides a substantial advance for RNA structure modeling, laying a strong foundation for the development of future RNA secondary structure prediction algorithms.

Langeberg CJ
Kim T
Nagle R
Meredith C
Garuadapuri DA
Doudna JA
Cate JHD


Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

Abstract: RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on S. pyogenes Cas9 ( Spy Cas9) variants with wide-ranging genome editing efficiencies that differ in PAM binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.

Shi H
Al-Sayyad N
Wasko KM
Trinidad MI
Doherty EE
Vohra K
Boger RS
Colognori D
Cofsky JC
Skopintsev P
Bryant Z
Doudna JA


Abstract: RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.

CRISPR-Cas Systems
Gene Editing
RNA, Guide, CRISPR-Cas Systems
Streptococcus pyogenes
Humans
CRISPR-Associated Protein 9
HEK293 Cells
Bacterial Proteins
DNA
Nucleotide Motifs


Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis

Abstract: Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation1. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

Weiss T
Kamalu M
Shi H
Li Z
Amerasekera J
Zhong Z
Adler BA
Song MM
Vohra K
Wirnowski G
Chitkara S
Ambrose C
Steinmetz N
Sridharan A
Sahagun D
Banfield JF
Doudna JA
Jacobsen SE

Arabidopsis
Gene Editing
Plant Viruses
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems
Plants, Genetically Modified
Transgenes
Genome, Plant


Directed evolution expands CRISPR-Cas12a genome editing capacity

Abstract: CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.


A miniature CRISPR-Cas10 enzyme confers immunity by an inverse signaling pathway

Abstract: Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signaling that share mechanistic features with human anti-viral systems 1 . In these pathways, including CBASS and type III CRISPR systems in bacteria and cGAS-STING in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response 2-4 . In a surprising inversion of this process, we show here that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that maintains cell health. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TMβ. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TMβ-mediated cell death due to inner membrane collapse. These findings unveil a powerful new defense strategy against virus-mediated immune suppression, expanding our understanding of oligonucleotides in cell health and disease. These results raise the possibility of similar protective roles for cyclic oligonucleotides in other organisms including humans.

Doherty EE
Adler BA
Yoon PH
Hsieh K
Loi K
Armbuster EG
Lahiri A
Bolling CS
Wilcox XE
Akkati A
Iavarone AT
Pogliano J
Doudna JA


Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection

Abstract: Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA. We show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. Early phage genes are expressed from the EPI vesicle, demonstrating its functionality as a prokaryotic, transcriptionally active, membrane-bound organelle. We also show that the phage nucleus is essential, with genome replication beginning after the injected DNA is transferred from the EPI vesicle to the phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

Armbruster EG
Rani P
Lee J
Klusch N
Hutchings J
Hoffman LY
Buschkaemper H
Enustun E
Adler BA
Inlow K
VanderWal AR
Hoffman MY
Daksh D
Aindow A
Deep A
Rodriguez ZK
Morgan CJ
Ghassemian M
Laughlin TG
Charles E
Cress BF
Savage DF
Doudna JA
Pogliano K
Corbett KD
Villa E
Pogliano J

Genome, Viral
Organelles
Viral Proteins
Bacteriophages
Virus Replication
Cell Nucleus


Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates

Abstract: The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.

Aris KDP
Cofsky JC
Shi H
Al-Sayyad N
Ivanov IE
Balaji A
Doudna JA
Bryant Z

CRISPR-Cas Systems
CRISPR-Associated Proteins
DNA, Superhelical
R-Loop Structures
RNA, Guide, CRISPR-Cas Systems
Kinetics
DNA
Endodeoxyribonucleases
Bacterial Proteins


Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing

Abstract: Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy, we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least 2-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing.

Karp H
Zoltek M
Wasko K
Vazquez AL
Brim J
Ngo W
Schepartz A
Doudna JA

Ribonucleoproteins
Gene Editing
CRISPR-Cas Systems
Humans
Electroporation
HEK293 Cells
CRISPR-Associated Protein 9


CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Abstract: Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.

Adler BA
Al-Shimary MJ
Patel JR
Armbruster EG
Colognori D
Charles EJ
Miller KV
Lahiri A
Cui ML
Oromí-Bosch A
Voelker A
Trinidad M
Lee J
Beurnier S
Boger R
Nomburg J
Barrangou R
Mutalik VK
Schoeniger JS
Pogliano JA
Savage DF
Doudna JA
Cress BF

Bacteriophages
Genomics
CRISPR-Cas Systems
Genome, Viral
Phylogeny
RNA-Binding Proteins
Genetic Fitness
Bacteriophage lambda


Mutational scanning of TnpB reveals latent activity for genome editing

Abstract: TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the protein's mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

Thornton BW
Weissman RF
Rodriguez JE
Tran RV
Duong BT
Terrace CI
Nagalakshmi U
Austin G
Groover ED
Wang FZ
Park JU
Georgieva V
Tartaglia J
Cho MJ
Dinesh-Kumar SP
Doudna JA
Savage DF


Single-molecule live-cell RNA imaging with CRISPR-Csm

Abstract: Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual native NOTCH2 and MAP1B transcripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation of NOTCH2 mRNA at the endoplasmic reticulum and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Xia C
Colognori D
Jiang XS
Xu K
Doudna JA

Clustered Regularly Interspaced Short Palindromic Repeats


Mechanism-guided engineering of a minimal biological particle for genome editing

Abstract: The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.

Ngo W
Peukes J
Baldwin A
Xue ZW
Hwang S
Stickels RR
Lin Z
Satpathy AT
Wells JA
Schekman R
Nogales E
Doudna JA

Gene Editing
Humans
CRISPR-Cas Systems
Lentivirus
Ribonucleoproteins
HEK293 Cells
CRISPR-Associated Protein 9
Genetic Vectors


Functional protein mining with conformal guarantees

Abstract: Molecular structure prediction and homology detection offer promising paths to discovering protein function and evolutionary relationships. However, current approaches lack statistical reliability assurances, limiting their practical utility for selecting proteins for further experimental and in-silico characterization. To address this challenge, we introduce a statistically principled approach to protein search leveraging principles from conformal prediction, offering a framework that ensures statistical guarantees with user-specified risk and provides calibrated probabilities (rather than raw ML scores) for any protein search model. Our method (1) lets users select many biologically-relevant loss metrics (i.e. false discovery rate) and assigns reliable functional probabilities for annotating genes of unknown function; (2) achieves state-of-the-art performance in enzyme classification without training new models; and (3) robustly and rapidly pre-filters proteins for computationally intensive structural alignment algorithms. Our framework enhances the reliability of protein homology detection and enables the discovery of uncharacterized proteins with likely desirable functional properties.

Boger RS
Chithrananda S
Angelopoulos AN
Yoon PH
Jordan MI
Doudna JA

Proteins
Algorithms
Computational Biology
Protein Conformation
Data Mining
Databases, Protein
Reproducibility of Results


CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation

Abstract: RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.

Soczek KM
Cofsky JC
Tuck OT
Shi H
Doudna JA

CRISPR-Cas Systems
CRISPR-Associated Proteins
DNA
Base Pairing
RNA, Guide, CRISPR-Cas Systems
Endodeoxyribonucleases
Bacterial Proteins
Nucleic Acid Conformation
Gene Editing
Cryoelectron Microscopy
Models, Molecular


RNA language models predict mutations that improve RNA function

Abstract: Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. RNA structure prediction is not yet possible due to a lack of high-quality reference data associated with organismal phenotypes that could inform RNA function. We present GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences to experimental and predicted optimal growth temperatures of GTDB reference organisms. Using GARNET, we develop sequence- and structure-aware RNA generative models, with overlapping triplet tokenization providing optimal encoding for a GPT-like model. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identify mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

Shulgina Y
Trinidad MI
Langeberg CJ
Nisonoff H
Chithrananda S
Skopintsev P
Nissley AJ
Patel J
Boger RS
Shi H
Yoon PH
Doherty EE
Pande T
Iyer AM
Doudna JA
Cate JHD

Mutation
Escherichia coli
Nucleic Acid Conformation
RNA
RNA, Ribosomal
Ribosomes
Deep Learning


SARS-CoV-2 evolution balances conflicting roles of N protein phosphorylation

Abstract: All lineages of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, contain mutations between amino acids 199 and 205 in the nucleocapsid (N) protein that are associated with increased infectivity. The effects of these mutations have been difficult to determine because N protein contributes to both viral replication and viral particle assembly during infection. Here, we used single-cycle infection and virus-like particle assays to show that N protein phosphorylation has opposing effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation, such as Delta (N:R203M), Iota (N:S202R), and B.1.2 (N:P199L). A new open reading frame encoding a truncated N protein called N*, which occurs in the B.1.1 lineage and subsequent lineages of the Alpha, Gamma, and Omicron variants, supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection.

Syed AM
Ciling A
Chen IP
Carlson CR
Adly AN
Martin HS
Taha TY
Khalid MM
Price N
Bouhaddou M
Ummadi MR
Moen JM
Krogan NJ
Morgan DO
Ott M
Doudna JA

SARS-CoV-2
Phosphorylation
Humans
Virus Replication
Evolution, Molecular
COVID-19
Coronavirus Nucleocapsid Proteins
Phosphoproteins
Virus Assembly
Mutation
Vero Cells
Chlorocebus aethiops
Animals


Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein

Abstract: Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.

Chen K
Han H
Zhao S
Xu B
Yin B
Lawanprasert A
Trinidad M
Burgstone BW
Murthy N
Doudna JA

Gene Editing
Animals
CRISPR-Cas Systems
Ribonucleoproteins
Mice
Nanoparticles
Liver
Lung
CRISPR-Associated Protein 9
Humans
Lipids
Liposomes


Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex

Abstract: Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

Tuck OT
Adler BA
Armbruster EG
Lahiri A
Hu JJ
Zhou J
Pogliano J
Doudna JA

Bacteriophages
Cryoelectron Microscopy
Deoxyribonucleases
Dimerization
DNA Damage
DNA Helicases
Escherichia coli
Escherichia coli Proteins


Author Correction: Birth of protein folds and functions in the virome

Nomburg J
Doherty EE
Price N
Bellieny-Rabelo D
Zhu YK
Doudna JA


Impact of Severe Postoperative Multi-Organ Dysfunction on Long-Term Outcomes After Modified Morrow Surgery

Abstract: The modified Morrow surgery is the gold standard surgical treatment for hypertrophic obstructive cardiomyopathy (HOCM). While effective, it carries a risk of postoperative severe multi-organ dysfunction and its impact on long-term cardiovascular outcomes remains poorly understood and controversial. This observational cohort study included 380 HOCM patients undergoing the modified Morrow surgery between July 2015 and March 2025. Severe multi-organ dysfunction was defined as a maximum Sequential Organ Failure Assessment (SOFA) score ≥ 11. The primary endpoint was the long-term incidence of major adverse cardiac and cerebrovascular events (MACCEs). Secondary endpoints included heart failure hospitalization (HFH), postoperative atrial fibrillation (AF) ablation, and cardiovascular mortality. Compared to patients without severe multi-organ dysfunction (SOFA Maximum < 11, n = 234), those with SOFA Maximum ≥ 11 (n = 146) had significantly higher rates of MACCEs (53.4% vs. 17.5%, p < 0.001), cardiovascular mortality (11.6% vs. 0.9%, p < 0.001), HFH (21.9% vs. 8.5%, p < 0.001), and postoperative AF ablation (17.8% vs. 3.8%, p < 0.001). Multivariable Cox proportional hazards regression demonstrated that severe multi-organ dysfunction was independently associated with cardiovascular mortality (HR = 4.157, p = 0.047), MACCEs (HR = 2.296, p < 0.001), HFH (HR = 1.834, p = 0.042), and postoperative AF ablation (HR = 3.987, p < 0.001). Other independent predictors included family history of hypertrophic cardiomyopathy, left ventricular end-systolic dimension, postoperative left ventricular outflow tract velocity, and postoperative surgical intensive care unit ventilation time. Postoperative severe multi-organ dysfunction is independently associated with an increased risk of adverse long-term outcomes after modified Morrow surgery. Incorporating the SOFA score into postoperative risk stratification may help identify high-risk patients warranting intensified long-term management.

Wang T
Ta L
Zhang K
Dong R
Xiao Y
Wang J

Humans
Female
Male
Cardiomyopathy, Hypertrophic
Multiple Organ Failure
Postoperative Complications
Time Factors
Risk Factors
Cardiac Surgical Procedures
Treatment Outcome
Aged
Middle Aged
Incidence
Retrospective Studies
Follow-Up Studies
Severity of Illness Index


ORTO-Measured Orthorexia Nervosa-Related Tendencies Among Dancers Before and During the COVID-19 Pandemic: A Multivariate Analysis

Abstract: This study aimed to examine the changes in orthorexia nervosa (ON) tendencies among dancers at different levels before and during the COVID-19 pandemic. This retrospective repeated-measures observational study included 128 professional, semi-professional, and amateur dancers (86 females, 42 males; 19-40 years) from various dance styles. Orthorexia nervosa tendencies before and during COVID-19 were assessed using ORTO-11 and ORTO-15. Physical activity and anthropometric data were collected for both recalled pre-pandemic and pandemic periods, while energy and macronutrient intakes were assessed using a retrospective 24-h dietary recall. Data were analyzed using paired tests, effect sizes (ES), and multivariate linear regression. During the pandemic, dancers reported approximately 50% lower weekly dance frequency and daily dance duration than recalled pre-pandemic values, with large and significant effect sizes. Significant decreases were observed in ORTO-11 and ORTO-15 scores, indicating an increase in orthorexic tendencies. Pandemic-period self-reported body weight was higher than pre-pandemic body weight, whereas BMI did not differ significantly between the recalled pre-pandemic and pandemic-period assessments. In multivariate analyses, female sex remained an independent predictor for both ORTO-11 and ORTO-15 scores. Compared with recalled pre-pandemic scores, pandemic-period ORTO-11 and ORTO-15 scores were lower, suggesting higher ORTO-measured health-oriented eating tendencies among dancers. Given the retrospective self-report design and the methodological limitations of ORTO-based instruments, these findings should not be interpreted as evidence of clinical ON prevalence or precise longitudinal change. Nevertheless, these ORTO-measured tendencies appeared to differ not only behaviorally but also in relation to nutrient intake patterns. Female sex was associated with pandemic-period ORTO scores, but this finding should be interpreted cautiously because psychological, sociocultural, and clinical variables were not assessed.

Journal of human nutrition and dietetics : the official journal of the British Dietetic Association

Ozlu Karahan T
Kenger EB
Tekin D
Agopyan A

Humans
Dancing
Female
COVID-19
Retrospective Studies
Adult
Male
Feeding and Eating Disorders
Multivariate Analysis
Young Adult
Feeding Behavior
SARS-CoV-2
Pandemics
Exercise
Diet
Energy Intake


LC-OCT for Cutaneous Lupus Erythematosus: Characterization Across Disease Subtypes and Differentiation From Rosacea

Abstract: Cutaneous lupus erythematosus (CLE) encompasses a heterogeneous group of autoimmune skin manifestations that often overlap clinically with other inflammatory dermatoses, particularly rosacea. While histopathology remains the gold standard, its invasiveness limits routine and longitudinal use. Line-field confocal optical coherence tomography (LC-OCT) is an emerging non-invasive imaging modality capable of providing real-time, near-histological resolution of skin architecture. In this single-center cross-sectional observational study, 45 patients (30 CLE: 16 acute, 5 subacute, 9 chronic; 15 rosacea) underwent LC-OCT imaging of active lesions. Images were independently evaluated using predefined criteria reflecting histopathological features. LC-OCT identified key histopathological correlates of CLE in vivo. Interface dermatitis features (including dermoepidermal junction disruption, basal cell vacuolization and band-like inflammatory infiltrate) were highly prevalent in ACLE and SCLE, while chronic CLE was characterized by epidermal atrophy, hyperkeratosis, fibrosis and adnexal destruction. In contrast, rosacea exhibited a vascular and folliculocentric pattern with frequent peri-adnexal inflammation and Demodex infestation, without interface changes. Interobserver agreement was substantial to excellent for most criteria. In the ACLE versus rosacea comparison, interface dermatitis features like band-like DEJ infiltrate, DEJ disruption, basal vacuolization and apoptotic bodies were associated with ACLE. Conversely, rosacea exhibited a vascular-folliculocentric profile with peri-infundibular infiltrates and Demodex mites. In conclusion, LC-OCT enables non-invasive visualization of disease-specific microarchitectural patterns in CLE, reflecting both inflammatory and chronic remodelling changes. In this pilot study, it shows potential in differentiating CLE from rosacea and may support diagnosis, staging and treatment monitoring in clinical practice. Larger, multicenter studies are required to confirm the role of LC-OCT in the non-invasive diagnosis of CLE.

Traini DO
Palmisano G
Ventura R
Cannizzaro MV
De Simone C
Rizzuti P
Pacucci VA
Cerasuolo PG
Piunno S
Gavotti C
Vigilante M
Maccari A
Caldarola G
Ortolan A
Suppa M
Di Stefani A
Agostino MA
Peris K

Humans
Rosacea
Tomography, Optical Coherence
Lupus Erythematosus, Cutaneous
Female
Cross-Sectional Studies
Male
Adult
Middle Aged
Diagnosis, Differential
Skin
Aged


Microneedling-assisted Panax ginseng exosome protocol for vulvovaginal symptoms and vulvar skin tone changes: A preliminary pilot observational study

Abstract: BackgroundNonhormonal approaches for vulvovaginal symptoms and vulvar skin concerns are increasingly being explored. However, clinical evidence for combined microneedling-assisted topical protocols in this setting remains limited.ObjectivesThis preliminary pilot observational study explored the short-term feasibility, patient-reported outcomes, tolerability, and qualitative photographic findings associated with a combined microneedling-assisted Panax ginseng exosome protocol with adjunctive inner gel use.DesignSingle-arm preliminary pilot observational study.MethodsFourteen women (mean age, 48.6 years; range, 24-63 years) underwent three treatment sessions at 2-week intervals, followed by a final assessment 2 weeks after the third treatment session, resulting in a total study duration of 6 weeks from baseline to final follow-up for each participant. Microneedling was performed on the vulvar area using a 2-mm dermaroller, followed by topical application of a Panax ginseng exosome formulation. Participants also used an adjunctive inner gel between visits. Outcomes included the Vulvovaginal Symptom Questionnaire (VSQ), the Day-to-Day Impact of Vaginal Aging (DIVA) questionnaire, procedural pain assessed using a visual analogue scale (VAS), safety observations, and qualitative clinical photographic assessment.ResultsTotal VSQ score decreased after treatment (mean change, -2.86; 95% confidence interval [CI], -4.76 to -0.95; p = 0.00648). Total DIVA score also decreased (mean change, -5.43; 95% CI, -9.55 to -1.30; p = 0.0138), with significant improvement in the daily activities domain (median change, -2.0; p = 0.00849). The emotion and sexual life domains showed directional decreases but did not reach statistical significance. Procedure-related pain was transient and generally decreased within 30 minutes after treatment. Clinical photographs showed qualitative observational changes in pigmentation intensity and skin tone uniformity. No serious adverse events were observed during the short follow-up period.ConclusionThis small uncontrolled pilot study found that the combined microneedling-assisted Panax ginseng exosome protocol with adjunctive inner gel use was associated with short-term improvements in patient-reported vulvovaginal symptoms and qualitative vulvar skin tone observations. Because of the single-arm design, small sample size, combined intervention, subjective photographic assessment, and short follow-up, these findings should be interpreted as preliminary and hypothesis-generating.

Humans
Female
Pilot Projects
Middle Aged
Adult
Panax
Percutaneous Collagen Induction
Vulva
Young Adult
Vagina
Treatment Outcome
Skin Aging


Selinexor combined bortezomib, lenalidomide, and dexamethasone for newly diagnosed multiple myeloma with high-risk factors: a single-arm, multi-center, prospective observational clinical study

Abstract: This study aimed to evaluate the efficacy and safety of selinexor combined with bortezomib, lenalidomide, and dexamethasone (the XVRd regimen) for newly diagnosed multiple myeloma (NDMM) patients with high-risk features. This single-arm, open-label, prospective observational study recruited patients between August 2022 and November 2024. The study consisted of an induction phase (21-day cycles) and a maintenance phase (28-day cycles). During induction, all enrolled patients aged ≥18 years received the XVRd regimen containing oral selinexor 60 mg weekly. After induction, patients transitioned to maintenance therapy with selinexor plus lenalidomide. The primary endpoints were the overall response rate (ORR) and minimal residual disease (MRD) negative rate. Secondary endpoints included progression-free survival (PFS), treatment safety, and tolerability. A total of 30 patients were enrolled, with a median age of 62.1 years (range, 47-73 years). Twenty-four patients (80%) presented with extramedullary plasmacytoma or plasma cell leukemia (PCL). The ORR across the entire cohort was 93.3% (28/30), including 5 patients with stringent complete response (sCR), 9 with complete response (CR), 4 with very good partial response (VGPR), and 10 with partial response (PR). Five patients (16.7%) achieved MRD negativity after induction treatment. Survival analysis demonstrated a median PFS of 16.2 months (95% CI, 14.6-NA), a 1-year PFS rate of 89% (95% CI, 0.78-1), and unreached median overall survival (OS). The overall safety profile was manageable. The XVRd regimen may yield promising efficacy with a tolerable safety profile in NDMM patients with extramedullary disease (EMD) and/or high-risk cytogenetic abnormalities. Chinese Clinical Trial Registry, registration number: ChiCTR2200062860.

Yin J
Yu S
Wu H
Zhou X
Li X
Yuan C
Chu X
Wang B
Hao L
Wei L
Zhong Y

Antineoplastic Combined Chemotherapy Protocols

Journal Article
Observational Study
Multicenter Study


Movement Behaviors and Quality of Life in Cardiac Rehabilitation Programs: A Study Based on the Canadian 24-Hour Movement Guidelines

Abstract: The Canadian 24-Hour Movement Guidelines integrate recommendations on physical activity (PA), sedentary behavior (SB), and sleep. Individuals in cardiac rehabilitation programs (CRPs) have specific characteristics that may influence adherence to these 24-Hour Movement Guidelines and their relationship with quality of life. This study aimed to investigate the proportion of CRP participants who meet the recommendations of the Canadian 24-Hour Movement Guidelines and to evaluate the association between levels of PA, SB, and sleep duration with the quality-of-life domains of these individuals. This was a cross-sectional observational study including CRP participants (mean age: 68.46 ± 10.15 years; 58.1% with coronary artery disease). Quality of life was assessed using the 36-Item Short-Form Health Survey (SF-36). Movement behaviors (PA, SB, and sleep) were measured by accelerometers. Compliance with the guidelines was described using percentage values. Associations between PA, SB, and sleep with quality-of-life domains were analyzed using quantile regression. The significance level was set at 5%. Among the 105 participants analyzed, only 2.9% simultaneously met all three recommendations. Individual adherence was 29.5% (PA), 9.5% (SB), and 34.3% (sleep). A negative association was identified between SB and functional capacity, and positive associations were found between PA and the domains of functional capacity, vitality, and social aspects. Sleep duration was not associated with quality-of-life domains. Few CRP participants simultaneously meet all three recommendations of the Canadian 24-Hour Movement Guidelines, with partial adherence being more frequent. Higher levels of PA and lower time spent in SB were associated with better quality of life, whereas sleep duration showed no significant association.

Physiotherapy research international : the journal for researchers and clinicians in physical therapy

Soares JCÁ
Vigilato MEM
Valente HB
Moliterno AH
Silva JPLN
Christofaro DGD
Vanderlei LCM

Humans
Quality of Life
Cross-Sectional Studies
Female
Male
Aged
Cardiac Rehabilitation
Canada
Sedentary Behavior
Middle Aged
Sleep Duration
Sleep
Guideline Adherence
Exercise
Practice Guidelines as Topic