Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

doi:10.1016/j.molcel.2015.10.030

Cited by 103 publications

(128 citation statements)

References 25 publications

Supporting

Mentioning

117

Contrasting

Order By: Relevance

“…By contrast, a previously identified type I anti-CRISPR protein (AcrE2; (Pawluk et al, 2014)) did not associate with NmeCas9 (Figure 2A, Figure S2). In a parallel experiment, the anti-CRISPRs did not bind significantly to AnaCas9 (Figure 2B, Figure S2) (Jinek et al, 2014; Ma et al, 2015a), a distantly related type II-C Cas9 homolog with ~20% sequence identity to NmeCas9. These data demonstrate that the anti-CRISPRs identified in this study specifically bind to NmeCas9.…”

Section: Resultsmentioning

confidence: 97%

See 1 more Smart Citation

Naturally Occurring Off-Switches for CRISPR-Cas9

Pawluk

Amrani

Zhang

et al. 2016

Cell

353

409

View full text Add to dashboard Cite

Summary CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These “anti-CRISPRs” were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9), and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable “off-switches” for CRISPR-Cas9 activity, and provide a genetically-encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes.

show abstract

Section: Resultsmentioning

confidence: 97%

“…Purified Cas9:sgRNA was dialyzed into 20 mM HEPES pH 7.5, 250 mM NaCl, 5% glycerol, 1 mM DTT and 1 mM PMSF) for protein interaction experiments. 6xHis-MBP-tagged AnaCas9 was purified from E. coli Rosetta (DE3) cells as described previously (Ma et al, 2015a). …”

Section: Star Methodsmentioning

confidence: 99%

Naturally Occurring Off-Switches for CRISPR-Cas9

Pawluk

Amrani

Zhang

et al. 2016

Cell

353

409

View full text Add to dashboard Cite

show abstract

“…There also may be other Acrs that have converged on this mechanism. Second, type IIC Cas9 orthologs are capable of tracrRNA- and PAM-independent DNA cleavage of single stranded DNA catalyzed by the HNH-domain (Ma et al, 2015; Zhang et al, 2015). In addition to inhibition of double-stranded DNA cleavage, AcrIIC1 would also be able to inhibit this single-stranded cleavage activity, whereas inhibitors of PAM binding such as AcrIIA4 may not be able to.…”

Section: Discussionmentioning

confidence: 99%

A Broad-Spectrum Inhibitor of CRISPR-Cas9

Harrington¹,

Doxzen²,

Ma³

et al. 2017

Cell

Self Cite

218

317

View full text Add to dashboard Cite

SUMMARY 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.

show abstract

“…Some enzymes simply fail to work and some choose their substrates promiscuously, necessitating thorough biochemical characterization [59][60][61][62][63][64][65] . Nevertheless, orthologous Cas9 enzymes with different PAM requirements increase targeting flexibility and allow multiplexing for combinatorial genetic screens, as demonstrated for SpCas9 and SaCas9 6,11,66 .…”

Section: Article Preprintmentioning

confidence: 99%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

View full text Add to dashboard Cite

The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

show abstract

Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes

Cited by 103 publications

References 25 publications

Naturally Occurring Off-Switches for CRISPR-Cas9

Naturally Occurring Off-Switches for CRISPR-Cas9

A Broad-Spectrum Inhibitor of CRISPR-Cas9

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Contact Info

Product

Resources

About