ABSTRACT
Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.
Subject(s)
CRISPR-Cas Systems , DNA, Single-Stranded , DNA, Single-Stranded/genetics , Genome , CRISPR-Associated Protein 9/genetics , Epigenome , Gene Editing/methodsABSTRACT
The ability to analyze the transcriptomic and epigenomic states of individual single cells has in recent years transformed our ability to measure and understand biological processes. Recent advancements have focused on increasing sensitivity and throughput to provide richer and deeper biological insights at the cellular level. The next frontier is the development of multiomic methods capable of analyzing multiple features from the same cell, such as the simultaneous measurement of the transcriptome and the chromatin accessibility of candidate regulatory elements. In this chapter, we discuss and describe SHARE-seq (Simultaneous high-throughput ATAC, and RNA expression with sequencing) for carrying out simultaneous chromatin accessibility and transcriptome measurements in single cells, together with the experimental and analytical considerations for achieving optimal results.
