Systematicin vitrospecificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Abstract

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

CRISPR-Cas effector specificity and target mismatches determine phage escape outcomes

“…PAM-distal crRNA mismatches did not influence the location of phage target mutations under pressure from Cas9. Accordingly, previous work found little to no synergistic effect of pairs of mismatches in the PAM distal region on Cas9 cleavage 69 . The enrichment of PAM-distal mutations after exposure to Cas12a and not Cas9 may also be influenced by their cut sites, which are in the PAM-distal region and downstream of the target for Cas12a and in the seed region for Cas9.…”

An efficient and cost-effective purification protocol for Staphylococcus aureus Cas9 nuclease

“…Purified SaCas9 can be directly used for in vitro applications. The new methodology is superior to the majority of conventional approaches, which rely on expensive infrastructure ( E.g., French press, high frequency sonicator, or fast protein liquid chromatography), 5 , 6 , 7 , 8 and thereby rendering SaCas9 production more cost-efficient. The average cost for the production of 10 mg of SaCas9 proteins is 86.36 USD ( Table 1 ), significantly lesser than several commercial sources, without compromising the quality of the enzyme itself.…”

“… 3 , 4 However, acquiring this protein remains a challenge for many laboratories that are not adequately equipped for protein purification. 5 , 6 , 7 , 8 , 9 For instance, fast protein liquid chromatography was used for purifying Cas9 proteins in several studies and the equipment requires a hefty initial investment. 6 , 7 , 8 Here, we report an advancement of the purification methods for SaCas9 from bacterial cells.…”

“… 5 , 6 , 7 , 8 , 9 For instance, fast protein liquid chromatography was used for purifying Cas9 proteins in several studies and the equipment requires a hefty initial investment. 6 , 7 , 8 Here, we report an advancement of the purification methods for SaCas9 from bacterial cells. A major advantage of this methodology is to achieve over 90% purity, at large batch sizes (concentrations at 1 mg/L) within a day.…”

CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives

“…Among the few other options available for generating base pair changes, mutation locations could only be determined by site but not the precise base 50 . The mutation frequencies of different bases within the mutation window are different and might be biased when applied to a genome-wide screen 51 . Also, the base editing protein complex is substantial, requiring efficient intracellular delivery tools, and its function may be impaired in some dense genome structures 52 .…”