Landscape of target:guide homology effects on Cas9-mediated cleavage

Abstract

To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in ‘seed’ sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13–18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9–gRNA interaction and cleavage beyond the general paradigm of site determination based on the ‘seed’ sequence and PAM.

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

“…A complementary approach is to generate a synthetic library that more thoroughly covers the space of possible off-target sites. To date, such studies have primarily been performed in vitro 17,18 and in yeast 19 . While specificity of the Cas9 from S. pyogenes has been previously profiled in human cells via cell surface marker knockout and flow cytometry 20 , the pairwise library approach described here provides an important alternative method.…”

Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

“…Cas9 dosage can also affect the kinetics and modulate specificity, both in vitro (Pattanayak et al, 2013; Fu et al, 2014a) and in cells (Hsu et al, 2013). For example, by plasmid transfection, a 5-fold drop in dose led to a 7-fold increase in the specificity ratio at the cost of a ~2-fold decrease in on-target efficiency, emphasizing the importance of titrating delivery conditions to optimize specificity for any given application (Hsu et al, 2013).…”

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

“…This approach would have several benefits over the traditional methodologies currently in trial as it does not rely on viral delivery, does not involve long-term expression off a nucleic acid cassette, and has low rates of off-target editing (Kim et al, 2014; Schumann et al, 2015). As Cas9 technology is further developed, the efficiency and off-target rate should improve, making these advantages even more stark (Doench et al, 2016; Fu et al, 2014; Kleinstiver et al, 2016; Slaymaker et al, 2016). …”