Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Abstract

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.

Fig. 1:. MRE11 ChIP molecularly characterizes Cas-induced DSBs.

(A) DISCOVER-Seq workflow. (B) DNA repair proteins assemble at a Cas9-induced DSBs. ChIP-Seq tracks for DNA repair proteins in K562 cells edited with VEGFA (+) or non-targeting gRNA (−). (C) Zoomed in data from (B). (D) ChIP-qPCR dynamics of repair protein binding and indel formation (also Fig. S2). (E) Coverage and reads for MRE11 ChIP-Seq at the VEGFA on-target (also Fig. S3B–C). Most reads end in the Cas9 cutsite. (F) Aggregate reads over multiple on- and off-target sites binned into gRNA binding the sense (n=7) or antisense (n=7) strand reveal asymmetric MRE11 recruitment consistent with in vitro models (schematic) (10). (G) MRE11 ChIP-Seq in K562 cells edited with AsCas12a-RNP visualizes multiple overhangs produced by Cas12a (12).

Fig. 2.. Unbiased off-target discovery using DISCOVER-Seq.

(A) Off-target sequences identified with DISCOVER-Seq and indel frequencies for VEGFA_site2 in K562s in one representative replicate. NC: non-coding. n.d.: not determined due to difficulties in PCR amplification. (B) and (C) Sequences, DISCOVER scores and indel frequencies for RNF2 and HBB gRNAs in K562s. (D) DISCOVER scores and indel frequencies are correlated (Spearman correlation) (also Fig. S9–10). On-target site in red.

Fig. 3.. DISCOVER-Seq in patient-derived iPSCs and in vivo.

(A) CMT patient-derived iPSCs were edited with HiFi-RNPs targeting the wild-type or mutant allele. (B) Patient-specific HSPB1 alleles. (C) DISCOVER scores and indel frequencies of off-targets for HSPB1 WT- and Mut- gRNAs. (D) Allele specificity of WT- and Mut-gRNAs by dropout amplicon-NGS indicate allele-specificity for the Mut-gRNA but not the WT-gRNA. (E) DISCOVER-Seq reads contain identifiable WT or Mut sequences distinguishing allele-specificity. (F) On-target (liver) and off-target (lung) tissues for DISCOVER-Seq (n=2 each) and amplicon-NGS (n=3) were harvested at indicated times after adenoviral injection. (G) DISCOVER-Seq distinguishes nuclease activity in target and non-target tissues at the on-target locus. (H) DISCOVER scores and indel frequencies by amplicon-NGS or ICE (*) for on-and off-targets in mouse livers. Black diamonds: not characterized by VIVO (14). NC: non-coding; n.d.: not determined.