Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Abstract

Background One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. Results The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Conclusions Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

CROP: a CRISPR/Cas9 guide selection program based on mapping guide variants

“…Running GUIDE-seq and CIRCLE-seq for in vivo and in vitro off-target measurement, respectively, would set a good benchmark for this. Additionally, a new more affordable technique for in vitro off-target measurement using Nanopore sequencing technology (Nano-OTS) 24 could also be used to provide similar data for future experiments. In conclusion, CROP can be readily downloaded from GitHub server ( https://github.com/vaprilyanto/crop ) and used as stand-alone software in any currently available computers to provide gRNA off-target propensity.…”

Rgen-Seq for Highly Sensitive Amplification-Free Screen of Off-Target Sites of Gene Editors

“…Given this assumption, one would expect on average an even read coverage on both sides of the cut. However, recent genome-wide screening of Cas9-generated breaks by PacBio's and ONT's nanopore real time sequencing 33 methods revealed highly asymmetrical read coverage of the PAM proximal and distal regions. Figure 2b shows the coverage of the PAM proximal and distal regions is skewed in RGEN-seq too but can be leveled if the end repair (ER) step is done along with A-tailing.…”

Hypoxia-directed tumor targeting of CRISPR/Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles

“…To overcome this limitation, many efforts have focused on targeting unique sequence variants that are found only in cancer cells, such as viral and fusion oncogenes (37)(38)(39)(40), minimizing the risk of any potential toxicity in normal tissue. Nevertheless, in addition to the largely limited number of targets that can be pursued with this approach, there is also a growing concern about the risks of off-target mutations that can arise with unchecked expression of the Cas9 throughout the body (41)(42)(43). Based on these concerns, we set out to evaluate the potential of an HRE-directed CRISPR/Cas9 to specifically induce cancer cell death in hypoxia.…”