Protocol for the functional evaluation of genetic variants using saturation genome editing
Open accessAbstract
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
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MAVEs in Hap1 cells have been semi-automated for large-scale deployment in functional genomics.
“Together, these factors have led to MAVEs in Hap1 cells being semi-automated for large-scale deployment in functional genomics (74).”
From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [74]
The ‘Before you begin’ section describes saturation genome editing in HAP1-A5 cells to study variant effects at scale, and ‘Expected outcomes’ describes high-throughput mutational data. The copy does not say the workflow has been semi-automated for large-scale deployment; a listed automated cell counter is optional and does not establish that claim. (LLM, conf=0.86)
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Cites
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Cited by
Sorted by citations
Massively parallel functional genomic assays in endocrinology: from promise to delivery
Xu, J., Aslanzadeh, V., Kudla, G., & Semple, R. K. (2026) · Journal of Molecular Endocrinology · doi:10.1530/jme-25-0134
- Authors
- Obolenski, S., Olvera-León, R., Sun, D., Adams, D. J., & Waters, A. J.
- Affiliations
- Leiden University Medical Center (NL)
- Wellcome Sanger Institute (GB)
- Corresponding author
- Adams, David J.
- Waters, Andrew J.
- Venue
- STAR Protocols
- Publisher
- Elsevier BV
- Published
- 2025-06
- Volume
- 6
- Issue
- 2
- Pages
- 103710
- ISSN
- 2666-1667 (ISSN-L)
- PMC
- PMC12008576
- OpenAlex
- W4409255349
- Cited by
- 3
- References
- 2
- Citations by year
- 2026: 1 · 2025: 2
- FWCI
- 0.83
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- STAR Protocols (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Leiden Repository (Leiden University) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Cancer Research UK
- Wellcome Trust (220540/Z/20/A)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- saturation genome editing, CRISPR/Cas9, homology-directed repair, single guide RNA design, oligonucleotide primer design, single nucleotide variants
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Humans, Genetic Variation, Gene Library, High-Throughput Nucleotide Sequencing, CRISPR-Cas Systems
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