Abstract
BackgroundOne 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.ResultsThe 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.ConclusionsAmplification-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.
Highlights
One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA binding may induce off-target mutations
Two new amplification-free protocols for off-target sequencing We developed two methods for guide RNA (gRNA) off-target sequencing (OTS) (Fig. 1a, b)
SMRT-off-target sequencing” (OTS) is a modified version of a protocol we previously applied for detection of repeat expansions in human cell lines and blood samples [34, 35], while Nano-OTS is adapted from a targeted sequencing assay [36] used for detection of unknown fusion gene partners [37]
Summary
One ongoing concern about CRISPR-Cas genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. A major reason for caution is that the CRISPR-Cas system can induce mutations at locations other than the targeted site [9,10,11]. Development of more efficient and precise genome editing tools such as CRISPR-Cas12a [13] or prime-editing [14] could help alleviate some of the off-target concerns. Even with these new tools, off-target mutations cannot be excluded, in particular in cases where the DNA sequence of the cells subjected to genome editing is not completely known
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