CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets

Abstract

Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe CIRCLE-seq (Circularization for In vitro Reporting of CLeavage Effects by sequencing), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequence. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific SNPs, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides the most accessible, rapid and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9 described to date.

NmeCas9 is an intrinsically high-fidelity genome editing platform

“…Our ability to detect NTS25-OT1 677 editing with GUIDE-seq, despite its very low (0.06%) editing efficiency based on high-throughput 678 sequencing, indicates that our GUIDE-seq experiments can identify even very low-efficiency off-target 679 editing sites. As more off-target profiling strategies are developed that have ever-increasing sensitivities 680 (Cameron et al 2017;Tsai et al 2017;Yan et al 2017), it will be useful to test whether NmeCas9 off-681 targeting remains nearly always undetectable even with improved detection limits. 682…”

TEG-Seq: An Ion Torrent-Adapted NGS Workflow for in Cellulo Mapping of CRISPR Specificity

“…The data from our lab and others [28,31] were generated using either Ion-Torrent PGM or Illumina MiSeq, which yield approximately 15 million reads per run. The lower limit of detection using the PGM was approximately 110 RPM.…”

“…The marked DSB site is then amplified and sequenced using next-generation sequencing (NGS). Several in vitro methods including BLESS-seq [26], HTGTS [27] and CIRCLE-seq [28] have been developed for off-target detection. These methods, by nature of the fact that the genomic DNA substrate has been removed from a cellular context and stripped of all protein, tend to identify all possible on- and off-target cleavage sites for a particular gRNA.…”

“…We also developed a 96-well format workflow that enables the study of multiple samples in parallel. The sensitivity of this method reached the level of Targeted Amplicon-seq, which has been widely used to validate the known and predicted gRNA off-target sites through direct amplification and NGS [28,31]. …”

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

“…WGS of individual induced pluripotent stem cells clones reveals a large number of indels in the genome that are not the result of Cas9 activity, but rather a consequence of clonal variation or technical artefacts [57]. To circumvent these limitations, several methods have recently been developed to measure Cas9 off-target activity across the genome such as BLESS (labeling of DSBs followed by enrichment and sequencing), HTGTS (high-throughput genome-wide translocation sequencing), GUIDE-Seq (genome-wide unbiased identification of DSBs enabled by sequencing), Digenome-Seq ( in vitro Cas9-digested WGS) [58], IDLV (detection of off-targets using integrase-deficient lentiviral vectors) [59] and most recently, SITE-Seq (a biochemical method that identifies DNA cut sites) [60] and CIRCLE-Seq (an in vitro method for identifying off-target mutations) [61]. Overall, these unbiased methods tend to be less sensitive and have a lower throughput than biased targeted sequencing, in addition to typically requiring higher sequencing coverage and much more complex protocols.…”