Towards Therapeutic Applications of CRISPR­Cas Nucleases: Developing Technologies for in Vivo Gene Editing & Evaluating Genome- Wide Specificity

Abstract

Treating of genetic disease by using programmable nucleases to directly alter the genome have gained momentum following recent advances in genome editing technologies based on CRISPR-Cas RNA-guided DNA endonucleases. However, there exist key challenges that limit the in vivo application of CRISPR-Cas nucleases. Here, we present technical advances in improving the delivery and understanding the genome-wide specificity of CRISPR-Cas nucleases. For the former, the size of the commonly-used Cas9 from Streptococcus pyogenes (SpCas9) limits its ability to be delivered in many in vivo genome editing contexts. We characterize a smaller Cas9 from Staphylococcus aureus (SaCas9) that is ~1kb smaller in size than SpCas9 and thus can be readily packaged along with its RNA guide into the highly versatile adeno-associated virus (AAV) delivery vehicle for efficient genome editing in somatic tissue. Since in vivo applications also require a versatile method of measuring nuclease specificity in both treated cells and tissues, we initially evaluated the specificity of Cas9 by identifying DNA double strand breaks (DSBs) using Breaks Labeling, Enrichment on Streptavidin, and Sequencing (BLESS). Though we successfully identified on and off-target sites with base pair resolution, the technical challenges of BLESS motivated the development of an improved assay, Breaks Labeling In Situ and Sequencing (BLISS) that is more sensitive, higher throughput, and more quantitative in measuring CRISPR-Cas nuclease off targets. The development of an ‘all-in-one’ AAV-SaCas9 vector for high efficiency in vivo genome editing, with subsequent evaluation of nuclease specificity directly in the treated cells and tissues by direct DSB labeling using BLISS, provide key components of a toolkit for in vivo genome editing and help pave the way towards therapeutic strategies using CRISPR-Cas nucleases.