enAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9

Abstract

While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug's target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.

CRISPR-based therapeutics: current challenges and future applications

“…Combining Cas9 with Cas12, a more compact version of Cas9 recognizing a different PAM site (i.e., TTTV), may improve gRNA coverage of CRISPR mutagenesis scanning. An evolved version of Cas12, recognizing PAM sites beyond TTTV, employed for CRISPR mutagenesis screens uncovered new drug resistance mutations against inhibitors of NAMPT and KIF11 [45]. Evolved variants of Cas9 and Cas12 with broadened PAM compatibility may also address the coverage issue [46,47].…”

Enhanced single‐base mutation diversity by the combination of cytidine deaminase with DNA‐repairing enzymes in yeast

“…As CRISPR/Cas techniques continue to advance, a multitude of CRISPR-based targeted mutagenesis systems have emerged, including CHAnGE, MAGESTIC, and many others. [10][11][12][13] In these systems, Cas9 variants are capable of precisely identifying the targeted gene, while gRNAs facilitate accurate and traceable editing of the gene. This leads to the production of numerous mutants with varying genotypes.…”