Protocol for the functional evaluation of genetic variants using saturation genome editing

STAR Protocols, vol. 6, pp. 103710

Abstract

Saturation genome editing (SGE) employs CRISPR-Cas9 and homology-directed repair (HDR) to introduce exhaustive nucleotide modifications at specific genomic sites in multiplex, enabling the functional analysis of genetic variants while preserving their native genomic context. Here, we present a protocol for SGE-based variant evaluation in HAP1-A5 cells. We describe the steps for designing variant libraries, single-guide RNAs (sgRNAs), and oligonucleotide primers for PCR. We also detail the sample preparation before the SGE screen, the cellular screening process, and subsequent next-generation sequencing (NGS) library preparation. For complete details on the use and execution of this protocol, please refer to Radford et al., 1 Waters et al., 2 and Olvera-León et al. 3 • SGE protocol in HAP1-A5 cells to assess variant effects on cell fitness over time • Description of SGE library and sgRNA design, cloning, and HAP1-A5 cell culture • Steps for SGE screen and SGE-edited gDNA library preparation for NGS • SGE approach generates functional scores for all SNVs and key variants in CDS, intron, and UTRs Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Saturation genome editing (SGE) employs CRISPR-Cas9 and homology-directed repair (HDR) to introduce exhaustive nucleotide modifications at specific genomic sites in multiplex, enabling the functional analysis of genetic variants while preserving their native genomic context. Here, we present a protocol for SGE-based variant evaluation in HAP1-A5 cells. We describe the steps for designing variant libraries, single-guide RNAs (sgRNAs), and oligonucleotide primers for PCR. We also detail the sample preparation before the SGE screen, the cellular screening process, and subsequent next-generation sequencing (NGS) library preparation.

Authors 5

1. Sofia Obolenski Aachen

   Leiden University · Leiden University Medical Center · Wellcome Sanger Institute

   Affiliation as printed

   Experimental Cancer Genetics, Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA Cambridge, UK; Department of Dermatology, Leiden University Medical Centre, 2333 ZA Leiden, the Netherlands

2. Rebeca Olvera-León

   Wellcome Sanger Institute

   Affiliation as printed

   Experimental Cancer Genetics, Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA Cambridge, UK

3. Dijue Sun

   Wellcome Sanger Institute

   Affiliation as printed

   Experimental Cancer Genetics, Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA Cambridge, UK

4. David J. Adams corresponding

   Wellcome Sanger Institute

   Affiliation as printed

   Experimental Cancer Genetics, Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA Cambridge, UK. Electronic address: da1@sanger.ac.uk

5. Andrew J. Waters corresponding

   Wellcome Sanger Institute

   Affiliation as printed

   Experimental Cancer Genetics, Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA Cambridge, UK. Electronic address: aw28@sanger.ac.uk