CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering

Figure 6

Genome-wide methods for the detection of off-target sites caused by Cas9 nuclease. (A) dCas9-Chip-seq: in dCas9-Chip-seq, the sgRNA and catalytically dead Cas9 (dCas9) plasmid are transfected into the cells. dCas9 proteins bound to DNA are immunoprecipitated after the cross-linking and shearing. The immunoprecipitated dCas9-associated DNA is analyzed by HTGS. (B) GUIDE-seq: in GUIDE-seq, the DSBs generated by RGN in living cells are tagged by integration of a blunt, short, double-stranded oligodeoxynucleotide (dsODN) followed by unbiased tag amplification and high-throughput sequencing for mapping the off-target cleavage sites. Integration sites are identified by LAM-PCR and high-throughput sequencing. (C) IDLV capture: after the transfection of Cas9-sgRNA complexes, the IDLV particles are delivered to get integrated into the RGN induced DSBs. Integration sites are identified by LAM-PCR and high-throughput sequencing. (D) BLESS: in BLESS, the RGN induced DSBs are ligated with sequencing adapters followed by fragment enrichment and amplification for high-throughput sequencing. (E) HTGST: in HTGST, the RGN generated unknown “prey” sequences are captured by known “bait” sequences by end joining repair of DSBs. The captured bait sequences are subjected to LAM-PCR followed by high-throughput sequencing.