RESUMEN
Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.
Asunto(s)
Proteínas Asociadas a CRISPR , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/metabolismoRESUMEN
Prokaryotes use clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) proteins as an adaptive immune system. CRISPR-Cas systems preserve molecular memories of infections by integrating short fragments of foreign nucleic acids as spacers into the host CRISPR array in a process termed 'adaptation'. Functional spacers ensure a robust immune response by Cas effectors, which neutralizes subsequent infection through RNA-guided interference pathways. In this review, we summarize recent discoveries that have advanced our understanding of adaptation, with a focus on how functional spacers are generated and incorporated through many widespread, but type-specific, mechanisms. Finally, we highlight future directions and outstanding questions for a more thorough understanding of CRISPR adaptation.
Asunto(s)
Proteínas Asociadas a CRISPR , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-CasRESUMEN
CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.
Asunto(s)
Bacteriófagos , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Bacteriófagos/genética , ADN/genética , ARN/genética , Mutación/genéticaRESUMEN
CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Bacteriano/genética , Escherichia coli/genética , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/inmunología , Proteína 9 Asociada a CRISPR/aislamiento & purificación , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/inmunología , Proteínas Asociadas a CRISPR/metabolismo , ADN Bacteriano/inmunología , ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Complejos Multienzimáticos , Conformación de Ácido Nucleico , Conformación Proteica , Subunidades de Proteína , Especificidad por SustratoRESUMEN
Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.
CRISPR-Cas systems are commonly used for biotechnology. Their specificity has been studied extensively and has previously been thought to be well understood. In this work, we asked a simple question about the effect of metal ion concentration on CRISPR specificity; the results are surprising and striking. At the actual metal ion concentrations found in cells, Cas12a specificity is inverted in comparison to the higher metal ion conditions that are typically used in test-tube assays. The specificity observed at lower metal ion concentration is more relevant under cellular conditions.
RESUMEN
CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Bases de Datos Genéticas , Endodesoxirribonucleasas , Sistemas CRISPR-Cas/genética , Filogenia , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/clasificación , Proteínas Asociadas a CRISPR/genética , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/clasificación , Endodesoxirribonucleasas/genética , Enciclopedias como AsuntoRESUMEN
In this issue, Swarts et al. (2017) discover that prokaryotic Argonaute proteins generate their own DNA guide strands through a newly characterized, unguided "chopping" activity. Double-stranded chopping products are selectively loaded onto Argonaute, enabling DNA-guided defense against foreign DNA.
Asunto(s)
Proteínas Argonautas/genética , Células Procariotas , ADNRESUMEN
CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/genética , Edición Génica/métodos , Genoma , Bacterias/genética , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Bacteriófagos/crecimiento & desarrollo , Endonucleasas/química , Endonucleasas/clasificación , Endonucleasas/metabolismo , Ingeniería Genética , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Transcripción GenéticaRESUMEN
CRISPR-Cas adaptive immune systems uptake short "spacer" sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn2+ dependent, Cas1 preferentially uses Mg2+ over Mn2+. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Proteínas Asociadas a CRISPR/metabolismo , ADN/química , Endonucleasas/genética , Endonucleasas/metabolismo , Integrasas/genéticaRESUMEN
During type I-E CRISPR-Cas immunity, the Cascade surveillance complex utilizes CRISPR-derived RNAs to target complementary invasive DNA for destruction. When invader mutation blocks this interference activity, Cascade instead triggers rapid primed adaptation against the invader. The molecular basis for this dual Cascade activity is poorly understood. Here we show that the conformation of the Cse1 subunit controls Cascade activity. Using FRET, we find that Cse1 exists in a dynamic equilibrium between "open" and "closed" conformations, and the extent to which the open conformation is favored directly correlates with the attenuation of interference and relative increase in priming activity upon target mutation. Additionally, the Cse1 L1 motif modulates Cascade activity by stabilizing the closed conformation. L1 mutations promote the open conformation and switch immune response from interference to priming. Our results demonstrate that Cascade conformation controls the functional outcome of target recognition, enabling tunable CRISPR immune response to combat invader evolution.
Asunto(s)
Proteínas Asociadas a CRISPR/inmunología , Sistemas CRISPR-Cas/inmunología , Escherichia coli K12/inmunología , Proteínas de Escherichia coli/inmunología , Regulación Bacteriana de la Expresión Génica , Plásmidos/metabolismo , Sitios de Unión , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Carbocianinas/química , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/inmunología , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Mutación , Plásmidos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Coloración y Etiquetado/métodosRESUMEN
Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Staphylococcus aureus/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Edición Génica , Especificidad por SustratoRESUMEN
Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.
Asunto(s)
Acidaminococcus/enzimología , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/genética , Endodesoxirribonucleasas/metabolismo , Francisella/enzimología , Acidaminococcus/genética , Acidaminococcus/metabolismo , Proteínas Bacterianas/genética , Disparidad de Par Base , Secuencia de Bases , Proteínas Asociadas a CRISPR/genética , ADN/metabolismo , División del ADN , Endodesoxirribonucleasas/genética , Francisella/genética , Francisella/metabolismo , Edición Génica/métodos , Expresión GénicaRESUMEN
In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary "nonself" DNA sequences for destruction, while avoiding binding to "self" sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for nonself target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved three base pair motif that is required for nonself target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade.
Asunto(s)
Proteínas de Escherichia coli/fisiología , Escherichia coli/inmunología , Subunidades de Proteína/fisiología , Sitios de Unión , ADN/química , Escherichia coli/genética , Escherichia coli/virología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/inmunología , Modelos Inmunológicos , Modelos Moleculares , Conformación de Ácido Nucleico , Subunidades de Proteína/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Bacteriano/fisiología , Ribonucleoproteínas/química , Ribonucleoproteínas/inmunología , Ribonucleoproteínas/fisiologíaRESUMEN
CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naïve' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Asociadas a CRISPR/metabolismo , Endodesoxirribonucleasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , MutaciónRESUMEN
The U2/U6 snRNA complex is a conserved and essential component of the active spliceosome that interacts with the pre-mRNA substrate and essential protein splicing factors to promote splicing catalysis. Here we have elucidated the solution structure of a 111-nucleotide U2/U6 complex using an approach that integrates SAXS, NMR, and molecular modeling. The U2/U6 structure contains a three-helix junction that forms an extended "Y" shape. The U6 internal stem-loop (ISL) forms a continuous stack with U2/U6 Helices Ib, Ia, and III. The coaxial stacking of Helix Ib on the U6 ISL is a configuration that is similar to the Domain V structure in group II introns. Interestingly, essential features of the complex--including the U80 metal binding site, AGC triad, and pre-mRNA recognition sites--localize to one face of the molecule. This observation suggests that the U2/U6 structure is well-suited for orienting substrate and cofactors during splicing catalysis.
Asunto(s)
Conformación de Ácido Nucleico , ARN de Hongos/química , ARN Nuclear Pequeño/química , Saccharomyces cerevisiae/genética , Resonancia Magnética Nuclear BiomolecularRESUMEN
Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.
RESUMEN
CRISPR-Cas adaptive immune systems uptake short 'spacer' sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn 2+ dependent, Cas1 preferentially uses Mg 2+ over Mn 2+ . The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.
RESUMEN
The efficiency of targeted DNA insertion by CRISPR transposons is improved.
Asunto(s)
Sistemas CRISPR-Cas , Elementos Transponibles de ADN , Edición Génica , Transposasas , Edición Génica/métodosRESUMEN
CRISPR-associated (Cas) endonucleases specifically target and cleave RNA or DNA based on complementarity to a guide RNA. Cas endonucleases - including Cas9, Cas12a, and Cas13 - have been adopted for a wide array of biotechnological tools, including gene editing, transcriptional modulation, and diagnostics. These tools are facilitated by ready reprogramming of guide RNA sequences and the varied nucleic acid binding and cleavage activities observed across diverse Cas endonucleases. However, the large size of most Cas endonucleases (950-1400 amino acids) can restrict applications. The recent discovery of miniature Cas endonucleases (400-800 amino acids) provides the potential to overcome this limitation. Here, we review recent advances in understanding the structural mechanisms of two miniature Cas endonucleases, Cas12f and Cas12j.
Asunto(s)
Sistemas CRISPR-Cas , Endonucleasas , Endonucleasas/química , ADN/genética , ARN/metabolismo , Aminoácidos/metabolismoRESUMEN
The response of bacteria to the threat posed by phages depends on their local environment.