Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9

Sounak Sahu; Teresa Sullivan; Eileen Southon; Dylan Caylor; Josephine Geh; Shyam K Sharan

doi:10.1016/j.xpro.2023.102702

Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9

STAR Protoc. 2023 Dec 15;4(4):102702. doi: 10.1016/j.xpro.2023.102702. Epub 2023 Nov 9.

Authors

Sounak Sahu¹, Teresa Sullivan², Eileen Southon², Dylan Caylor², Josephine Geh², Shyam K Sharan³

Affiliations

¹ Mouse Cancer Genetics Program, Centre for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA. Electronic address: [email protected].
² Mouse Cancer Genetics Program, Centre for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
³ Mouse Cancer Genetics Program, Centre for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA. Electronic address: [email protected].

Abstract

Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.¹.

Keywords: Bioinformatics; CRISPR; Cancer; Genetics; Genomics; Molecular Biology.

MeSH terms

CRISPR-Cas Systems* / genetics
DNA
Gene Editing / methods
Genomics
RNA, Guide, CRISPR-Cas Systems*

Substances

RNA, Guide, CRISPR-Cas Systems
DNA