Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

Basgall, Erianna M.; Goetting, Samantha C.; Goeckel, Megan E.; Giersch, Rachael M; Roggenkamp, Emily; Schrock, Madison N.; Halloran, Megan; Finnigan, Gregory C.

doi:10.1099/mic.0.000635

Cited by 104 publications

(109 citation statements)

References 45 publications

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“…In fact, proof-of-concept CRISPR-based homing gene drives have recently been developed in several organisms including yeast (29)(30)(31)(32), flies (33)(34)(35)(36)(37)(38), mice (39), and two malaria vector species, Anopheles gambiae (40)(41)(42) and Anopheles stephensi (43).…”

Section: Introductionmentioning

confidence: 99%

Development of a Confinable Gene-Drive System in the Human Disease Vector,Aedes aegypti

Li¹,

Yang²,

Kandul³

et al. 2019

Preprint

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Aedes aegypti, the principal mosquito vector for many arboviruses that causes yellow fever, dengue, Zika, and chikungunya, increasingly infects millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has recently sparked significant enthusiasm for the genetic control of mosquito populations, however no such system has been developed in Ae. aegypti. To fill this void and demonstrate efficacy in Ae. aegypti, here we develop several CRISPR-based split-gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, 2 mathematical models predict that these systems could spread anti-pathogen effector genes into wild Ae. aegypti populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effectorlinked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission. Significance StatementAe. aegypti is a globally distributed arbovirus vector spreading deadly pathogens to millions of people annually. Current control methods are inadequate and therefore new technologies need to be innovated and implemented. With the aim of providing new tools for controlling this pest, here we engineered and tested several split gene drives in this species. These drives functioned at very high efficiency and may provide a tool to fill the void in controlling this vector. Taken together, our results provide compelling path forward for the feasibility of future effector-linked split-drive technologies that can contribute to the safe, sustained control and potentially the elimination of pathogens transmitted by this species.

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Section: Introductionmentioning

confidence: 99%

Development of a Confinable Gene-Drive System in the Human Disease Vector,Aedes aegypti

Li¹,

Yang²,

Kandul³

et al. 2019

Preprint

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“…A number of Acrs have been identified for type I [23,[25][26][27][28], type II [29][30][31][32] and type V [28,33] CRISPR-Cas systems. Acrs can be adapted to regulate CRISPR-Cas activities in bacteria [34], yeast [35] and mammalian cells [29,31,34,[36][37][38]. Biosensor [39] and synthetic circuits [40] can be devised based on Acr-coupled CRISPR-Cas systems.…”

Section: Inhibition Off-target Activitymentioning

confidence: 99%

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

Cui

Wang

et al. 2019

Preprint

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Background CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system.Excessive and prolonged expression CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over the dimensions of time and dose has created the demand of developing CRISPR-Cas off-switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpyCas9) proteins in an allosteric manner. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpyCas9 while retaining its on-target activity. ConclusionG8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

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“…A recent approach provided a more detailed mechanism of the AcrIIA2-mediated inhibition of spCas9, showing that AcrIIA2 as a DNA mimic interacts with the PAM recognition residues of Cas9, thereby preventing target dsDNA detection [79]. Basgall et al [80] demonstrated that AcrIIA2 and AcrIIA4 are able to inhibit the functional activity of Cas9 in vivo within haploid yeast and in an active gene-driven system, providing the first documented use of AcrIIA2 and AcrIIA4 to inhibit such a system. Furthermore, AcrIIA2 has been shown to exhibit temperature-dependent anti-CRISPR inhibitory activity, which may explain why AcrIIA2 can only partially inhibit spCas9 activity in E. coli and human cells [81].…”

Section: Type II Anti-crispr Proteinsmentioning

confidence: 99%

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

2019

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Clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas adaptive immune defense systems, which are widely distributed in bacteria and Archaea, can provide sequence‐specific protection against foreign DNA or RNA in some cases. However, the evolution of defense systems in bacterial hosts did not lead to the elimination of phages, and some phages carry anti‐CRISPR genes that encode products that bind to the components mediating the defense mechanism and thus antagonize CRISPR‐Cas immune systems of bacteria. Given the extensive application of CRISPR‐Cas9 technologies in gene editing, in this review, we focus on the anti‐CRISPR proteins (Acrs) that inhibit CRISPR‐Cas systems for gene editing. We describe the discovery of Acrs in immune systems involving type I, II, and V CRISPR‐Cas immunity, discuss the potential function of Acrs in inactivating type II and V CRISPR‐Cas systems for gene editing and gene modulation, and provide an outlook on the development of important biotechnology tools for genetic engineering using Acrs.

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Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

Cited by 104 publications

References 45 publications

Development of a Confinable Gene-Drive System in the Human Disease Vector,Aedes aegypti

Development of a Confinable Gene-Drive System in the Human Disease Vector,Aedes aegypti

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

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

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