Targeted sequencing of potential CRISRP/Cas9 off-target editing sites in modified human CD34+ Haematopoietic Stem Cells. Homo sapiens

We report the use of short biotinylated oligonucleotides for the targeted pulldown of potential CRISPR/Cas9 off-target loci in condition and control to measure the frequency of off target editing Overall design: NG Capture-C was used to investigate the Potential off-target CRISPR/Cas9 loci. NG Capture-C combines 3C library preparation with oligonucleotide capture for the desired viewpoint restriction fragments. Potential off-target CRISPR/Cas9 loci were identified using the Sanger tool (http://www.sanger.ac.uk/htgt/wge/find_off_targets_by_seq) and 70bp oligonucleotides designed for target-pulldown. Whole genome amplified (WGA) DNA from edited clones from two editing strategies was pooled and indexed. Indexed WGA DNA was then pulled down in two rounds of hybridization with biotinylated oligonucleotides and streptavadin beads to enrich for putative off-target editing loci.