RESUMEN
CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.
Asunto(s)
Proteína 9 Asociada a CRISPR , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Edición Génica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , ADN/metabolismo , ADN/genética , ADN/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Edición Génica/métodos , Cinética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismoRESUMEN
RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Microscopía por Crioelectrón , Bacterias/metabolismo , ARN/metabolismo , ADN/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismoRESUMEN
The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Elementos Transponibles de ADN , Deinococcus , Endodesoxirribonucleasas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/química , ADN/genética , ADN/metabolismo , ADN/ultraestructura , Elementos Transponibles de ADN/genética , ARN Guía de Sistemas CRISPR-Cas/química , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/ultraestructura , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/ultraestructura , Deinococcus/enzimología , Deinococcus/genética , Especificidad por SustratoRESUMEN
CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.
Asunto(s)
Proteínas Asociadas a CRISPR , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/genética , Plásmidos , ARN , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from Francisella novicida and the type V-B system from Alicyclobacillus acidoterrestris. In contrast to other class 2 CRISPR-Cas systems, we found that for the type V-A and V-B systems, the Cas12 nucleases are dispensable for spacer acquisition, with only Cas1 and Cas2 (type V-A) or Cas4/1 and Cas2 (type V-B) being necessary and sufficient. Whereas the catalytic activity of Cas4 is not essential for adaptation, Cas4 activity is required for correct protospacer adjacent motif selection in both systems and for prespacer trimming in type V-A. In addition, we provide evidence for acquisition of RecBCD-produced DNA fragments by both systems, but with spacers derived from foreign DNA being incorporated preferentially over those derived from the host chromosome. Our work shows that several spacer acquisition mechanisms are conserved between diverse CRISPR-Cas systems, but also highlights unexpected nuances between similar systems that generally contribute to a bias of gaining immunity against invading genetic elements.
Asunto(s)
Proteínas Asociadas a CRISPR , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , ADN , Endonucleasas/genética , Edición GénicaRESUMEN
Genome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Reparación del ADN/genética , Endodesoxirribonucleasas/genética , Edición Génica , Proteína 9 Asociada a CRISPR/genética , Escherichia coli/genética , Proteínas Luminiscentes/genética , Plásmidos/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
Over the last decade, research on distinct types of CRISPR systems has revealed many structural and functional variations. Recently, several novel types of single-polypeptide CRISPR-associated systems have been discovered including Cas12a/Cpf1 and Cas13a/C2c2. Despite distant similarities to Cas9, these additional systems have unique structural and functional features, providing new opportunities for genome editing applications. Here, relevant fundamental features of natural and engineered CRISPR-Cas variants are compared. Moreover, practical matters are discussed that are essential for dedicated genome editing applications, including nuclease regulation and delivery, target specificity, as well as host repair diversity.
Asunto(s)
Sistemas CRISPR-Cas/genética , Exorribonucleasas/genética , Edición Génica , Exorribonucleasas/metabolismoRESUMEN
Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.