340. Development of a Nuclease Screen to Improve Cas9 Targeting Specificity

Abstract

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) bacterial immunity targets invading nucleic acid sequences using an RNA guide (gRNA). The well-defined Cas9 from Streptococcus pyogenes’ type II CRISPR system has been utilized as a gene editing tool in a vast assortment of organisms. However, an inherent lack of specificity limits the potential of the current system and presents challenges to interpreting experimental results. A plasmid cleavage based screening platform has been developed with the intent to increase the fidelity of the Cas9 system. Utilizing both positive and negative selection, the screen can be used to identify variants of Cas9 or its gRNA with improved specificity. Positive selection of variants with activity towards the appropriate target site proceeds through the removal of a plasmid containing an inducible suicide gene. Conversely, negative selection of variants with reduced activity on off-target substrates involves the retention of a plasmid with an antibiotic resistance gene. Preliminary results suggest that a gRNA with an insertion in the scaffold region of the RNA can decrease the level of off-target cleavage. These results suggest that the screening platform has the potential to identify novel protein or RNA variants with greater targeting specificity compared to the naturally occurring components. A Cas9 system with enhanced fidelity will expand the potential applications of the technology and accelerate its ability to interrogate biological systems.