High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

By Yanfang Fu, Jennifer A. Foden, Cyd Khayter, Morgan L. Maeder, Deepak Reyon, Keith Joung, Jeffry D. Sander. 2013

3,275 citations recorded by the source corpus.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell–based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

Subfields: Molecular Biology

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Citations in the corpus

Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites (2015, background): “However, RGENs can also cleave off-target DNA sites with several mismatches (Cho et al., 2014; Fu et al., 2013) or with missing or additional nucleotides (Lin et al., 2014), compared to gRNA sequences.”
RNA-guided genome editing in plants using a CRISPR-Cas system. (2013, background): “Most recently, the off-target issue associated with the CRISPR–Cas9 system has been addressed using the human cell system and a number of studies demonstrated that mismatches at the 3′ end of gRNA seeds drastically reduce the off-target effect (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a).”
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA (2014, result): “This is consistent with previous reports that the 10–12 bp PAM-proximal “seed” region is critical for the Cas9-catalyzed DNA cleavage (Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jinek et al., 2012; Mali et al., 2013a; Pattanayak et al., 2013).”
In vivo genome editing using Staphylococcus aureus Cas9 (2015, background): “Although a number of studies have employed sequence similarity-based off-target search 22,26–30 or dCas9-ChIP 31,32 to predict off-target sites for Cas9, such approaches cannot assess the nuclease activity of Cas9 in a comprehensive and unbiased manner.”
Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition (2016, background): “CRISPR/Cas9 gene editing has been shown to generate offtarget mutations depending upon the experimental setting and cell type (35, 36).”
CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites (2014, neutral): “Conflict of interest: none declared.”
Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases (2014, background): “Very recently, three groups independently showed that RGENs indeed induce off-target mutations, even at sites that differ by 5 nt from on-target sites in human cells (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”