Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity

Abstract

The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.

Evolution of Resistance Against CRISPR/Cas9 Gene Drive

“…Since these alleles should carry similar fitness costs as the driver, they are not likely to rise in frequency. However, resistance may still evolve if some mutations change the target sequence without actually disrupting the gene, or if the ability to drive itself is already associated with some fitness costs, as will likely be the case if off-target effects of the driver have not been completely suppressed (Fu et al 2013;Anderson et al 2015;Zhang et al 2015). Another proposed strategy is the insertion of a new gene (e.g., a gene that prevents mosquitoes from transmitting malaria).…”

Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

“…3a). When performing this analysis, it is important to remember that Cas9 can potentially also cut genes with a one base mismatch in the sequence, in particular if the mismatch is far from the PAM sequence, while for two mismatches the enzyme does not cut in approximately 96% of the cases24.…”

“…A recent report by Anderson et al . showed that 96% of the crRNAs with two mismatches were not functional24. For one mismatch the Cas9 can still work efficiently, in particular if the mismatch is far from the PAM-sequence.…”

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Genetic Engineering: Robotic Genetic Surgery

“…One of the major issues with CRISPR is the ability to ensure that only the target gene is edited and changes hazardous to the individual's health are not introduced throughout the genome. Unwanted off-target site mutations could invariably lead to further malignancies and undesirable phenotypes [25]. In a recent study by Huang et al researchers have demonstrated the ability to perform human embryo editing with CRISPR, potentially triggering ethical dilemmas across the community [26, 27].…”