Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6

Fuchsbauer, Olivier; Swuec, Paolo; Zimberger, Claire; Amigues, Béatrice; Levesque, Sébastien; Agudelo, Daniel; Duringer, Alexis; Chaves-Sanjuán, Antonio; Spinelli, S.; Rousseau, Geneviève M.; Velimirovic, Minja; Bolognesi, M.; Roussel, Alain; Cambillau, Christian; Moineau, Sylvain; Doyon, Yannick; Goulet, Adeline

doi:10.1016/j.molcel.2019.09.012

Cited by 52 publications

(55 citation statements)

References 89 publications

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“…These orthologs also differ in their sensitivity to anti-CRISPR proteins as St1Cas9 is inhibited by both AcrIIA5 and AcrIIA6, whereas SaCas9 can only be blocked by AcrIIA5 (Supplemental Fig. S4E,F; Hynes et al 2018;Fuchsbauer et al 2019;Garcia et al 2019). This comparison between St1Cas9 and SaCas9 suggests that they function orthogonally and could be used in a combinatorial manner.…”

Section: Engineering St1cas9 Variants To Expand Its Targeting Rangementioning

confidence: 97%

“…the natural diversity found within S. thermophilus strains results in true reprograming toward a distinct PAM as opposed to relaxing specificity. In addition, despite their sequence and structural conservation (Nishimasu et al 2015;Fuchsbauer et al 2019) St1Cas9 variants could not cleave at SaCas9 PAMs (NNGRRT; where R is A or G) in human cells, further highlighting their specificity (Supplemental Fig. S4A-D).…”

Section: Engineering St1cas9 Variants To Expand Its Targeting Rangementioning

confidence: 99%

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Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

et al. 2020

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Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

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Section: Engineering St1cas9 Variants To Expand Its Targeting Rangementioning

confidence: 97%

Section: Engineering St1cas9 Variants To Expand Its Targeting Rangementioning

confidence: 99%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

et al. 2020

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“…To date, inhibition of target binding is the prevalent strategy. Thirteen anti-CRISPR proteins interfere with target recognition and binding (type I-F AcrIF1, AcrIF2 and AcrIF10 [34][35][36][37]; type II-A AcrIIA2, AcrIIA4, AcrIIA5 and AcrIIA6 [38][39][40][41][42][43][44][45][46][47]; type II-C AcrIIC3, AcrIIC4 and AcrIIC5 [48][49][50][51]; type V-A AcrVA1, AcrV4A and AcrVA5 [52][53][54][55][56][57][58]), while only five-block target cleavage (type I-E AcrIE1 [29,59]; type III-B AcrIIIB1 [12]; type I-F AcrIF3 [26,27]; type II-C AcrIIC1 and AcrIIC3 [48][49][50]) ( Figure 1). Given that DNA binding is the ratelimiting step of Cascade and Cas9-mediated interference activities [60,61], altering this step is, therefore, an efficient way to inactivate CRISPR-Cas interference.…”

Section: Inhibition Of Crispr-cas Interferencementioning

confidence: 99%

“…AcrIIA6 and AcrVA4 both function as dimers that tightly associate with a mixed protein-RNA region distinct from the DNA-binding crevasse and catalytic domains [47,54,57,58]. However, regions of both subunits that compose the AcrIIA6 dimer form each binding interface, while every subunit of the AcrV4A dimer contains one binding interface ( Figure 2B).…”

Section: Allosteric Inhibition and Clustering Of Effector Complexesmentioning

confidence: 99%

“…These anti-CRISPR proteins perturb the conformational changes required to bind to target DNA, thus locking St1Cas9 and LbCas12a effector complexes in a nonfunctional state. AcrIIA6 impairs St1Cas9 conformational rearrangements associated with PAM binding, which leads to the inhibition of target DNA recognition and binding [47]. In contrast, AcrVA4 does not affect PAM binding but impairs LbCas12a dynamics and structural changes required for the R-loop formation and stabilization (glossary), which prematurely stops the hybridization between the cRNA and target DNA [57].…”

Section: Allosteric Inhibition and Clustering Of Effector Complexesmentioning

confidence: 99%

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Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

Hardouin

Goulet

2020

Biochemical Society Transactions

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Bacteriophages (phages) and their preys are engaged in an evolutionary arms race driving the co-adaptation of their attack and defense mechanisms. In this context, phages have evolved diverse anti-CRISPR proteins to evade the bacterial CRISPR–Cas immune system, and propagate. Anti-CRISPR proteins do not share much resemblance with each other and with proteins of known function, which raises intriguing questions particularly relating to their modes of action. In recent years, there have been many structure–function studies shedding light on different CRISPR–Cas inhibition strategies. As the anti-CRISPR field of research is rapidly growing, it is opportune to review the current knowledge on these proteins, with particular emphasis on the molecular strategies deployed to inactivate distinct steps of CRISPR–Cas immunity. Anti-CRISPR proteins can be orthosteric or allosteric inhibitors of CRISPR–Cas machineries, as well as enzymes that irreversibly modify CRISPR–Cas components. This repertoire of CRISPR–Cas inhibition mechanisms will likely expand in the future, providing fundamental knowledge on phage–bacteria interactions and offering great perspectives for the development of biotechnological tools to fine-tune CRISPR–Cas-based gene edition.

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