Target-Enriched GUIDE-seq (TEG-seq)

TEG-seq is comparable to GUIDE-seq². Both methods use protected short double strand DNAs to tag break sites in the genome. These tags are then used as universal primer sites for amplification and NGS mapping of the sequences flanking the break site. The TEG-seq protocol uses amplification and sequencing primer design that makes it specifically compatible with the Ion Torrent line of NGS platforms. Comparisons with data from Illiumina-based GUIDE-seq experiments suggest that both methods yield similar results (Tang et al. manuscript submitted). TEG-seq was performed by ThermoFisher. Briefly, 150,000 Neuro-2a mouse cells (ATCC® CCL131™, derived from the A/J strain²⁴) were transfected with Cas9 protein (1µg) and a full-length synthetic Pnpla3 sgRNA (10 pmol, Synthego) complexed as ribonucleoprotein (RNP) along with a dsTag (1.25 pmol) using the Neon electroporation system, incubated for 3 days after which genomic DNA was isolated (PureLink® Genomic DNA, ThermoFisher) and subjected to the TEG-seq/GUIDE-seq procedure. Synthetic sgRNA was used for TEG-seq as we have found it to be more efficient than in vitro transcribed sgRNA for RNP electroporation. Amplicon reads were aligned to the reference mouse genome (GRCm38/mm10). The mapped reads were further preceded through Motif-Search, a plugin software for off-target search and read count (ThermoFisher). For easier comparison, total reads from the different samples were normalized by using Reads Per Million (RPM): total reads from the sample multiplied by one million and divided by total number of mapped reads of the NGS run. The off-target candidates with RPM > 1 were subjected to Targeted Amplicon re-sequencing (Ampli-seq). Primers flanking the cleavage sites and used for Ampli-seq are listed in Supplementary Table 2. Off-targets were confirmed by detection of either the dsTag or presence of indels >3 bp in the expected position upstream of the PAM site.