Abstract
CRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm off-target cleavage that cannot be detected by targeted deep sequencing in cells before. Second, we use this approach to detect multiple alternative PAMs recognized by SpCas9. These noncanonical PAMs are associated with low cleavage activity, but targets associated with these PAMs must be considered as potential off-target sites. Taken together, the GFP activation assay is a powerful platform for DNA cleavage detection in cells.
Highlights
The RNA-guided CRISPR/Cas9 system can introduce desired mutations into the genome and has a broad range of research and medical applications (Cong et al, 2013; Hwang et al, 2013; Mali et al, 2013; Xie et al, 2017; Wang et al, 2019a)
The GFP Activation Assay for Validation of Off-Targets
A target sequence with a protospacer adjacent motif (PAM) is inserted between ATG start codon and GFP coding sequence, disrupting GFP expression by frameshift mutation
Summary
The RNA-guided CRISPR/Cas system can introduce desired mutations into the genome and has a broad range of research and medical applications (Cong et al, 2013; Hwang et al, 2013; Mali et al, 2013; Xie et al, 2017; Wang et al, 2019a) This system consists of a Cas nuclease and a guide RNA (gRNA), which forms a Cas9-gRNA complex, recognizing a target sequence (protospacer) with a downstream protospacer adjacent motif (PAM), and induces a site-specific double-strand break (DSB) (Jinek et al, 2012; Cong et al, 2013; Mali et al, 2013). Off-target mutations can confound interpretation of the experiments and can have implications for the development of therapeutic applications
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