Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

By S. Cho, Sojung Kim, Yongsub Kim, Jiyeon Kweon, H. Kim, Sangsu Bae, Jin-Soo Kim. Genome Research, 2014

1,407 citations recorded by the source corpus.

RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

Subfields: Cardiology and Cardiovascular Medicine, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/9f9797e339c7cde8fbec533c2f068f415134e4e0

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In Cardiology and Cardiovascular Medicine

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Citations in the corpus

CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences (2014, positive): “Recent studies have shown that CRISPR / Cas9 systems non-specifically cleave genomic DNA sequences containing base-pair mis-matches (case a) generating off-target mutations in mammalian cells with considerable frequencies (19–24).”
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs (2014, background): “…nickases 8 ), in which two gRNAs targeted to adjacent sites on opposite DNA strands each recruit a Cas9 variant (Cas9-D10A) that nicks DNA instead of cutting both strands 7, 9, 10 , can reduce mutation frequencies at known off-target sites of single gRNA-guided Cas9 nuclease in human cells 7, 10 .”
Target specificity of the CRISPR-Cas9 system (2014, neutral): “It has also been reported that compared to in vivo conditions, Cas9 cutting is more promiscuous in vitro [43], i.”
Causal Structure Learning (2017, background): “If the gene knockout experiment has signiﬁcant oﬀ-target eﬀects (e.g., Cho et al., 2014), then this approach is problematic with respect to the autonomy assumption.”
Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing? (2019, background): “However, the majority of off-target sites exhibit mutation frequencies at least 2-fold lower than on-target mutation frequency if the off-target site has greater than one bp mismatch compared with the on-target site (Hsu et al., 2013; Pattanayak et al., 2013; Cho et al., 2014).”
GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases (2014, neutral): “In addition, chromosomal translocations can result from joining of on-and off-target RGN-induced cleavage events 8-11 .”
The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “One caveat to this approach is that two equally efficient gRNAs are needed to make an efficient nickase pair, potentially limiting the number of target sites given that not all gRNAs are equivalent in terms of activity (Cho et al., 2014).”
Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA[OPEN] (2015, background): “…various sites may yield different results, incorporation of the Cas9 D10A nickase (Gasiunas et al., 2012) may further improve gene editing options for Cas9, because the nickase version has been successfully applied to improve specificity (Mali et al., 2013; Cho et al., 2014; Fauser et al., 2014).”
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells (2014, neutral): “Some studies have shown that Cas9 is highly specific 21,30,31 , whereas other studies have demonstrated substantial Cas9 off-target activity 9,24,27,29,32 .”
Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides (2017, background): “Unfortunately, off-target DNA cleavage at sites that are highly homologous to on-target sites can lead to mutations at undesired genomic loci [5, 6] and to chromosomal rearrangements such as translocations and inversions [6–8].”