Benchmarking Nuclease Off-Target Effects

The top 10 most highly predicted loci for off-target nuclease activity of Cas9-3 and Cas12a-1 were identified by Benchling online software, while TALEN sites were predicted using PROGNOS (http://bao.rice.edu/cgi-bin/prognos/prognos.cgi) (Fine et al., 2014 (link)). PCR amplicons were designed to generate 150-200bp with the expected cut site in the centre. DNA from male healthy donor T cells edited with each nuclease platform (alongside untreated controls) was extracted at day 3 post nucleofection (Qiagen) and used as a template for on-target and off-target PCR reactions. The amplicons were then prepared for Illumina next generation sequencing by performing end repair, adapter ligation and bar coding using the NEBNext^® UltraTM II DNA Library Prep Kit (NEB) according to the manufacturer’s instructions. Libraries were quantified using the ddPCR™ Library Quantification Kit for Illumina TruSeq (Biorad), before sequencing using MiSeq Reagent Kit v2, 500cycles on an Illumina MiSeq platform (Illumina). The generated paired-end reads were processed using the command line version of the CRISPResso2 pipeline (Clement et al., 2019 (link)), obtained editing frequencies were compared to the untreated control samples using a one-sided Fisher’s exact test. *, **, and *** indicate p < 0.05, p < 0.01, p < 0.001, respectively.