In CRISPRs, an RGN from Streptococcus pyogenes (Cas9) is used. Cas9 requires a 20-nucleotide RNA guide (gRNA) sequence that is complementary to the target genomic DNA sequence where this sequence is adjacent to a NGG motif. The only other sequence requirement for CRISPR is that the first nucleotide of the gRNA is a G, but this is due to a requirement of the U6 promoter needed to express the RNA (Figure). Therefore, CRIPSRs provide a very flexible method to introduce double-stranded breaks into DNA. However, a central concern with gene editing techniques is the number of off-target double-stranded breaks that may be introduced.
This article* examines the specificity of the gRNAs used in CRISPR by monitoring the NHEJ-mediated repair of an EGFP-PEST reporter introduced as a single copy into cells. With this reporter gene assay, indel mutations can be monitored through the loss of GFP fluorescence.
In the study, three gRNAs were used against separate regions of the GFP gene. Variants of these gRNAs containing various nucleotide mismatches across the 20-nucleotide region were then constructed and tested by comparing the amount of GFP expression to wild-type sequences. This showed that mismatch tolerances depended on the target site, with site 2 being very intolerant to mutations in the 3′ end of the sequences, while sites 1 and 3 were tolerant to single-nucleotide mismatches except for a few sites at the 3′ end; the specific sites that were sensitive differed between target sites. Therefore, tolerances to mismatches were more sensitive at the 3′ end, consistent with previous reports, but the relative amount of tolerance was target site specific.
Next, the study looked for off-target mutations in endogenous genes. Six single gRNAs were used to target four different genes, and these were shown to efficiently introduce indel mutations into their target genes. However, examination of genomic loci that differed by one or two nucleotides (as well as genomic sites differing by three or six nucleotides, particularly in the 5′ end) showed high rates of off-target mutations. Rates of mutations at these sites for two of the four genes in U2OS, HEK293, and K562 cells ranged from a small percentage of the target gene to >125% of the target gene.
The rates of off-target mutations did not improve by expression of lower amounts of single gRNAs or Cas9. This article cautions that predicting such off-target mutations with CRISPR will not be easy because it appears to be target site specific, and this will be have to be carefully evaluated when constructing cell lines with this technology.