CRISPR‐Suppressor Scanning for Systematic Discovery of Drug‐Resistance Mutations

Abstract

CRISPR-Cas9 genome editing technologies have enabled complex genetic manipulations in situ, including large-scale, pooled screening approaches to probe and uncover mechanistic insights across various biological processes. The RNA-programmable nature of CRISPR-Cas9 greatly empowers tiling mutagenesis approaches to elucidate molecular details of protein function, in particular the interrogation of mechanisms of resistance to small molecules, an approach termed CRISPR-suppressor scanning. In a typical CRISPR-suppressor scanning experiment, a pooled library of single-guide RNAs is designed to target across the coding sequence(s) of one or more genes, enabling the Cas9 nuclease to systematically mutate the targeted proteins and generate large numbers of diverse protein variants in situ. This cellular pool of protein variants is then challenged with drug treatment to identify mutations conferring a fitness advantage. Drug-resistance mutations identified with this approach can not only elucidate drug mechanism of action but also reveal deeper mechanistic insights into protein structure-function relationships. In this article, we outline the framework for a standard CRISPR-suppressor scanning experiment. Specifically, we provide instructions for the design and construction of a pooled sgRNA library, execution of a CRISPR-suppressor scanning screen, and basic computational analysis of the resulting data.

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery

“…All sgRNAs meeting, these criteria were synthesized as an oligonucleotide pool (Twist Biosciences) and their sequences are listed in Supplementary file 1 . The sgRNA oligo pool was amplified, cloned into lentiCRISPRv2, and sequenced to confirm sgRNA representation as previously described ( Ngan et al, 2022 ; Joung et al, 2017 ; Canver et al, 2018 ). Lentivirus carrying the resulting pooled sgRNA tiling library was produced as described above and titered according to published procedure ( Ngan et al, 2022 ; Joung et al, 2017 ; Canver et al, 2018 ).…”

“…Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen). To measure the sgRNA composition of the population, the sgRNA expression cassette was PCR amplified using barcoded primers, purified, and sequenced as previously described ( Vinyard et al, 2019 ; Ngan et al, 2022 ; Joung et al, 2017 ; Canver et al, 2018 ). All samples were sequenced on a MiSeq (Illumina) using 150-cycle, single-end reads.…”

“…All samples were sequenced on a MiSeq (Illumina) using 150-cycle, single-end reads. Sufficient coverage of the sgRNA library was maintained in accordance with published recommendations ( Ngan et al, 2022 ; Joung et al, 2017 ; Canver et al, 2018 ).…”

“…Recently, we and others have used CRISPR–Cas9 tiling mutagenesis screens to uncover modes of small molecule action by identifying drug resistance mutations ( Neggers et al, 2018 ; Donovan et al, 2017 ; Vinyard et al, 2019 ; Gosavi et al, 2022 ; Kwok et al, 2022 ). In our approach, termed CRISPR-suppressor scanning, Cas9 is used to systematically mutate a target protein with a pooled library of single-guide RNAs (sgRNAs) spanning its entire coding sequence (CDS) to generate large numbers of diverse protein variants in situ ( Ngan et al, 2022 ). This surviving cellular pool is then treated with small molecule inhibitors to identify variants conferring drug resistance.…”