Systematic discovery of natural CRISPR-Cas12a inhibitors

Abstract

Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.

CasLocusAnno: a web‐based server for annotating cas loci and their corresponding (sub)types

“…Continuous works from scientific communities can enrich the overall number of Acr entries in public resources depending on emerging CRISPR-Cas tools [36,37]. Web-based servers [21,23] and standalone programs have been, in recent years, made available for CRISPR-Cas identification [22,35], nevertheless the constructed service and the developed standalone program can be still as complementary tools mainly because of the following reasons: Compared to other methods, CasLocusAnno facilitates selection of more reasonable Cas profile, because it chooses profiles with minimum e-values in each cluster. CasLocusAnno can also identify fused Cas proteins, whereas neither MAC-SYFINDER nor CRISPRCasFinder can do so because they select only profiles with the highest score.…”

Anti‐CRISPR proteins targeting the CRISPR‐Cas system enrich the toolkit for genetic engineering

“…In contrast to Cas9, Cas12a processes its own crRNAs, which have been exploited for multiplex genome editing, indicating the powerful potential of Cas12a in genome editing . Testing whether Cas12a inhibitors block or reduce Cas12a‐mediated genome editing in human cells has revealed that AcrVA1 provides the broadest inhibition of Cas12a . Moreover, we believe that newly discovered Acrs targeting type V CRISPR‐Cas systems will soon be used to precisely regulate Cas12a‐mediated gene editing and greatly promote the use of the CRISPR‐Cas system to better facilitate therapeutic genome editing and CRISPR disease diagnosis.…”

“…Furthermore, a homolog of Aca2 encoding a type II-C CRISPR-Cas system was found to be located upstream of the aca2 gene in strains of Neisseria meningitidis (N. meningitidis), and subsequent experiments revealed that the putative anti-CRISPR from B. oedipodis and a homolog from N. meningitidis inhibited the CRISPR-Cas9 system, demonstrating that Acrs are not limited to type I CRISPR-Cas systems [23]. Given that the Cas protein in B. oedipodis shares 47% amino acid sequence identity with the homolog in N. meningitidis, it was predicted that a putative Acr, named AcrIIC1, inhibits [20,89] the type II-C CRISPR-Cas system from N. meningitidis [23]. Moreover, continued iterative searches identified two other type II-C Acr family members in N. meningitidis, AcrIIC2 and AcrIIC3, but neither of these genes exhibits any similarity to the detectable sequence of AcrIIC1 [23].…”

“…Recently, Watters et al [20] identified three targeted Acrs (AcrVA1, AcrVA4, and AcrVA5) from M. bovoculi that inhibit or attenuate CRISPR-Cas12a (type V)-based genome editing in human cells. Marino et al [22] also concurrently reported the discovery of AcrVA1, AcrVA2, and AcrVA3 from M. bovoculi and confirmed that AcrVA1 inhibits a broad spectrum of Cas12a orthologs, including M. bovoculi Cas12a (MbCas12a), Acidaminococcus sp.…”

“…The first reported case of CRISPR protein inhibition in 2013 targeted the type I-F system from Pseudomonas aeruginosa (P. aeruginosa) [12], and the following year, four anti-CRISPR proteins (Acrs) targeting the type I-E systems were identified [13]. Subsequently, many types of type I Acr proteins have been systematically discovered, and because Acr research has proceeded quickly, recent studies have successively identified many Acr types that inhibit type I (I-E, I-F, I-D, I-C), type II (II-A, II-C), type III (III-B), and type V (Cas12a, also known as Cpf1) proteins [16][17][18][19][20][21][22][23][24]. Although the mechanism and function of Acrs have been systematically summarized by several recent reviews [25][26][27][28][29], their deeper and wider application as a potential tool for regulating CRISPR-Cas systems for gene editing deserves further attention from researchers.…”