Figure 7.

Off-target genome mapping. (a) Experimental schema for IDLV gene trapping. Nuclease mRNA for MT and TALEN and Cas9 mRNA and gRNA plasmid were introduced into Jurkats followed by transduction with an IDLV expressing GFP and puromycin. The IDLV is integrated into loci where a DNA break has occurred. LAM and nRLAM PCR experimental schema. LTR priming results in linear fragments that are converted to double-stranded DNA products that are barcoded, deep sequenced, and interrogated against the genome for off-target sites. (b) TRAC IDLV gene trapping confirmation at on-target TRAC locus. A PCR using LTR- and TRAC-specific primers (red and blue arrows shown in a) revealed presence of the IDLV cargo at the TRAC locus for all of the nuclease reagents visualized by agarose gel.