Abstract
Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of ‘anti-CRISPR’ proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing. One Cas9-based application of interest to the field of population control is that of the ‘gene drive’. Here, we demonstrate use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast. Furthermore, an unbiased mutational scan reveals that titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.
Highlights
The recent discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) system in prokaryotes [1] has led to a major shift in many fields of molecular biology and biotechnology [2,3,4]
Our study has demonstrated that the anti-CRISPR proteins AcrIIA2 and AcrIIA4 are able to inhibit the function of S. pyogenes Cas9 in vivo within haploid yeast and an active gene drive system
We found that epitopes or gene fusions were not tolerated on either terminus of AcrIIA2 in contrast to AcrIIA4
Summary
The recent discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) system in prokaryotes [1] has led to a major shift in many fields of molecular biology and biotechnology [2,3,4]. One particular arrangement of the Cas nuclease editing system has been recognized for its potential for pest management – namely, the ‘gene drive’. This genetic system has demonstrated the potential to combat insect-borne diseases such as malaria for its ability to rapidly impose population control through a super-Mendelian mechanism [11,12,13,14,15]. The Cas gene and its corresponding single guide (sgRNA) expression cassette are integrated at or in place of an endogenous locus; the nuclease is targeted to the WT copy of the deleted (or modified) native gene on the homologous chromosome within a diploid cell. There are still challenging technical (and ethical) hurdles facing the development and implementation of gene drive systems including (i) evolved resistance to artificial drive systems in native populations [14,15,16,17,18], (ii) biosecurity [19, 20] and (iii) public approval [21, 22]
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