Protocol for the functional evaluation of genetic variants using saturation genome editing
Sofia Obolenski1, Rebeca Olvera-León2, Dijue Sun2
1Experimental 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.
STAR Protocols
|April 8, 2025
Summary
Saturation genome editing (SGE) provides a method for multiplex nucleotide modification using CRISPR-Cas9 and homology-directed repair (HDR). This protocol details SGE for variant evaluation in HAP1-A5 cells, enabling functional genetic analysis.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Saturation genome editing (SGE) is a powerful technique for multiplex nucleotide modification.
- CRISPR-Cas9 and homology-directed repair (HDR) are key components of SGE.
- Functional analysis of genetic variants requires methods that preserve native genomic context.
Purpose of the Study:
- To present a detailed protocol for SGE-based variant evaluation.
- To enable the functional analysis of genetic variants in HAP1-A5 cells.
- To outline the steps for designing variant libraries, sgRNAs, and primers for PCR.
Main Methods:
- Design of variant libraries, single-guide RNAs (sgRNAs), and oligonucleotide primers for PCR.
- Sample preparation for SGE screening.
- Cellular screening and next-generation sequencing (NGS) library preparation.
Main Results:
- The protocol enables exhaustive nucleotide modifications at specific genomic sites.
- Multiplex editing allows for comprehensive functional analysis of genetic variants.
- The method preserves the native genomic context of evaluated variants.
Conclusions:
- This protocol provides a robust framework for SGE-based variant evaluation.
- The described methods facilitate the functional genomics of genetic variants.
- SGE is a valuable tool for understanding genetic variation and its functional impact.
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