Distinct patterns of Cas9 mismatch tolerancein vitroandin vivo

Abstract

Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

“…Only crRNAs unique within the human genome were used, with one unavoidable exception ( TOMM20 , where the locus sequence restricted crRNA choice), and crRNAs whose alternative binding sites include mismatches in the “seed” region and are in nongenic regions were prioritized whenever possible (Supplemental Figure S2; Graham and Root, 2015; Tsai et al. , 2015; Fu et al. , 2016).…”

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

“…Given their small size, DNA barcodes generally only minimally disrupt a locus but this needs to be verified for the locus of interest, as is the case for strategies involving targeting affinity handles by Cas9 or other approaches. Fortunately, genomic barcoding has come within reach for many research questions due to the availability emerging genome engineering strategies, in yeast and other organisms [63][64][65][66] . For example, in budding yeast, CRIPSR-Cas9 tools provide the means to integrate barcodes from simple oligonucleotide-derived repair templates with very high efficiency and precision without the need for a selectable marker.…”

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

“…We compared our kinetic model at saturating enzyme concentration to prior in vitro and cellular studies of wtCas9 and wtCas12a specificity Fu et al, 2016;Hsu et al, 2013;Kim et al, 2015Kim et al, , 2016Kim et al, , 2017aKleinstiver et al, 2016bKleinstiver et al, , 2016aPattanayak et al, 2013;Yan et al, 2017) (Figure 5E). Importantly, prior cellular high-throughput experiments enumerated off-target sites at multiple loci but at a single time point after transfection.…”