ABSTRACT
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
Subject(s)
CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cryoelectron Microscopy , DNA , Gene Editing , Humans , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , CRISPR-Associated Protein 9/metabolism , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , DNA/metabolism , DNA/genetics , Gene Editing/methods , Geobacillus stearothermophilus/genetics , Geobacillus stearothermophilus/metabolism , HEK293 Cells , Protein Domains , Genome, Human , Models, Molecular , Protein Structure, Tertiary , Nucleic Acid Conformation , Biocatalysis , Magnesium/chemistry , Magnesium/metabolismABSTRACT
CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.
Subject(s)
CRISPR-Cas Systems , Chromosome Aberrations , Gene Editing , T-Lymphocytes , Humans , Chromosomes , CRISPR-Cas Systems/genetics , DNA Damage , Gene Editing/methods , Clinical Trials as TopicABSTRACT
CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.
Subject(s)
Bacteriophages , CRISPR-Cas Systems , Animals , Humans , Cryoelectron Microscopy , Gene Editing , Genome , Bacteriophages/genetics , DNA , RNA , Mammals/geneticsABSTRACT
The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved â¼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , Cell Phone/instrumentation , Optical Imaging/methods , RNA, Viral/analysis , Viral Load/methods , Animals , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , CRISPR-Cas Systems , Cell Line , Coronavirus Nucleocapsid Proteins/genetics , Humans , Nasopharynx/virology , Optical Imaging/instrumentation , Phosphoproteins/genetics , Point-of-Care Testing , RNA Interference , RNA, Viral/genetics , Sensitivity and Specificity , Viral Load/economics , Viral Load/instrumentationABSTRACT
The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.
Subject(s)
CRISPR-Cas Systems/physiology , Clustered Regularly Interspaced Short Palindromic Repeats/physiology , Gene Editing/methods , CRISPR-Associated Proteins/chemistry , DNA/chemistry , Genome , Models, Molecular , RNA/chemistry , RNA, Guide, Kinetoplastida/geneticsABSTRACT
CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.
Subject(s)
CRISPR-Cas Systems , Endonucleases/antagonists & inhibitors , Viral Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriophages/genetics , Bacteriophages/metabolism , Endonucleases/chemistry , Endonucleases/genetics , Endonucleases/metabolism , Evolution, Molecular , HEK293 Cells , Humans , Protein Domains , Sequence AlignmentABSTRACT
Bacteria and archaea possess a range of defense mechanisms to combat plasmids and viral infections. Unique among these are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems, which provide adaptive immunity against foreign nucleic acids. CRISPR systems function by acquiring genetic records of invaders to facilitate robust interference upon reinfection. In this Review, we discuss recent advances in understanding the diverse mechanisms by which Cas proteins respond to foreign nucleic acids and how these systems have been harnessed for precision genome manipulation in a wide array of organisms.
Subject(s)
Archaea/genetics , Bacteria/genetics , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Genetic Engineering/methods , Animals , Archaea/immunology , Archaea/virology , Bacteria/immunology , Bacteria/virology , DNA, Viral/genetics , Endonucleases/chemistry , Endonucleases/genetics , Endonucleases/metabolism , Humans , Plants/geneticsABSTRACT
RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.
Subject(s)
RNA/analysis , CRISPR-Cas Systems , Cytoplasmic Granules/chemistry , Endonucleases/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Green Fluorescent Proteins/analysis , Humans , RNA, Guide, Kinetoplastida/analysis , RNA, Messenger/analysisABSTRACT
CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.
Subject(s)
Bacteriophage lambda/genetics , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , DNA Helicases/metabolism , DNA, Viral/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/virology , Escherichia coli/immunology , Escherichia coli/metabolism , Models, Biological , Repetitive Sequences, Nucleic AcidABSTRACT
Nineteen genetic therapies have been approved by the U.S. Food and Drug Administration (FDA) to date, a number that now includes the first CRISPR genome editing therapy for sickle cell disease, CASGEVY (exagamglogene autotemcel). This extraordinary milestone is widely celebrated because of the promise for future genome editing treatments of previously intractable genetic disorders and cancers. At the same time, such genetic therapies are the most expensive drugs on the market, with list prices exceeding $4 million per patient. Although all approved cell and gene therapies trace their origins to academic or government research institutions, reliance on for-profit pharmaceutical companies for subsequent development and commercialization results in prices that prioritize recouping investments, paying for candidate product failures, and meeting investor and shareholder expectations. To increase affordability and access, sustainable discovery-to-market alternatives are needed that address system-wide deficiencies. Here, we present recommendations of a multi-disciplinary task force assembled to chart such a path. We describe a pricing structure that, once implemented, could reduce per-patient cost tenfold and propose a business model that distributes responsibilities while leveraging diverse funding sources. We also outline how academic licensing provisions, manufacturing innovation and supportive regulations can reduce cost and enable broader patient treatment.
ABSTRACT
The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence1. Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database2,3. Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts. Structural comparisons suggested putative functions for up to 25% of unannotated viral proteins, including those with roles in the evasion of innate immunity. In particular, RNA ligase T-like phosphodiesterases were found to resemble phage-encoded proteins that hydrolyse the host immune-activating cyclic dinucleotides 3',3'- and 2',3'-cyclic GMP-AMP (cGAMP). Experimental analysis showed that RNA ligase T homologues encoded by avian poxviruses similarly hydrolyse cGAMP, showing that RNA ligase T-mediated targeting of cGAMP is an evolutionarily conserved mechanism of immune evasion that is present in both bacteriophage and eukaryotic viruses. Together, the viral protein structural database and analyses presented here afford new opportunities to identify mechanisms of virus-host interactions that are common across the virome.
ABSTRACT
Used widely for genome editing, CRISPR-Cas enzymes provide RNA-guided immunity to microbes by targeting foreign nucleic acids for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. These crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Nevertheless, Cas12c represses transcription and can defend bacteria against lytic bacteriophage infection when targeting an essential phage gene. Together, these results show that Cas12c employs targeted DNA binding to provide antiviral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity.
Subject(s)
Bacteriophages , CRISPR-Cas Systems , Antiviral Agents , Bacteria/genetics , Bacteriophages/genetics , Bacteriophages/metabolism , DNA/metabolism , Deoxyribonuclease I/metabolism , Deoxyribonucleases/genetics , Gene Expression , RNA/metabolismABSTRACT
A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.
Subject(s)
Gene Editing , RNA, Guide, Kinetoplastida , Animals , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Endonucleases/metabolism , Gene Editing/methods , Mammals/metabolism , RNA/genetics , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolismABSTRACT
RNA is involved in the regulation of multiple cellular processes, often by forming sequence-specific base pairs with cellular RNA or DNA targets that must be identified among the large number of nucleic acids in a cell. Several RNA-based regulatory systems in eukaryotes, bacteria and archaea, including microRNAs (miRNAs), small interfering RNAs (siRNAs), CRISPR RNAs (crRNAs) and small RNAs (sRNAs) that are dependent on the RNA chaperone protein Hfq, achieve specificity using similar strategies. Central to their function is the presentation of short 'seed sequences' within a ribonucleoprotein complex to facilitate the search for and recognition of targets.
Subject(s)
MicroRNAs/metabolism , RNA, Small Interfering/metabolism , Bacteria/genetics , CRISPR-Cas Systems , Gene Expression Regulation , Gene Silencing , MicroRNAs/genetics , RNA, Small Interfering/geneticsABSTRACT
Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.
Subject(s)
Nucleotidyltransferases/chemistry , Protein Engineering , Vibrio cholerae/enzymology , Amino Acid Sequence , Catalytic Domain , Humans , Immunity, Innate , Models, Molecular , Molecular Sequence Data , Nucleotidyltransferases/metabolism , Sequence Alignment , Substrate SpecificityABSTRACT
CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.
Subject(s)
Biocatalysis , CRISPR-Associated Proteins , CRISPR-Cas Systems , Genome, Bacterial , Integrases , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/metabolism , CRISPR-Associated Proteins/ultrastructure , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/immunology , Cryoelectron Microscopy , DNA/immunology , DNA/metabolism , Exonucleases/chemistry , Exonucleases/metabolism , Exonucleases/ultrastructure , Integrases/chemistry , Integrases/metabolism , Integrases/ultrastructure , Genome, Bacterial/geneticsABSTRACT
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
Subject(s)
Endodeoxyribonucleases/genetics , Escherichia coli/genetics , Gene Knockdown Techniques/methods , RNA Interference , Streptococcus pyogenes/enzymology , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Gene Expression , Streptococcus pyogenes/genetics , Transcription Elongation, Genetic , Transcription Initiation, Genetic , RNA, Small UntranslatedABSTRACT
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Subject(s)
Bacterial Proteins/genetics , Gene Targeting/methods , Streptococcus pyogenes , HEK293 Cells , HeLa Cells , Humans , Saccharomyces cerevisiae/genetics , RNA, Small UntranslatedABSTRACT
Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as 'Borgs'. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.