Saturation editing of genomic regions by multiplex homology-directed repair
Open accessAbstract
Saturation mutagenesis--coupled to an appropriate biological assay--represents a fundamental means of achieving a high-resolution understanding of regulatory and protein-coding nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or "safe-harbour" integration fail to capture the native context of the endogenous chromosomal locus. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage with multiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six-base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.
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Cas9 introduces a targeted double-strand break, which is repaired by homology-directed repair using donor templates, resulting in the incorporation of a single variant at the endogenous genomic locus.
“Cas9 introduces a targeted double-strand break, which is repaired by homology-directed repair using the donor templates, resulting in the incorporation of a single variant at the endogenous genomic locus (54, 55).”
From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [55]
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“Saturation genome editing (Findlay et al., 2014, 2018), which relies on homology-directed repair (HDR), has been used to screen all possible variants in several exons of BRCA1; while HDR approaches are effective in yeast (Sharon et al., 2018), the low efficiency of HDR in human cells has restricted…”
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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
- Findlay, G. M., Boyle, E. A., Hause, R. J., Klein, J. C., & Shendure, J.
- Affiliations
- University of Washington (US)
- Corresponding author
- Findlay, Gregory M.
- Shendure, Jay
- Venue
- Nature
- Published
- 2014-09
- Volume
- 513
- Issue
- 7516
- Pages
- 120-123
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4156553
- OpenAlex
- W2014747507
- Cited by
- 362
- References
- 8
- Citations by year
- 2026: 25 · 2025: 24 · 2024: 28 · 2023: 28 · 2022: 22 · 2021: 36 · 2020: 25 · 2019: 27 · 2018: 26 · 2017: 32 · 2016: 47 · 2015: 35 · 2014: 7
- FWCI
- 15.23
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- National Institutes of Health
- National Human Genome Research Institute (DP1HG007811)
- National Institute of General Medical Sciences (T32GM007266)
- National Center for Chronic Disease Prevention and Health Promotion (DP1 HG007811)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- saturation mutagenesis, genome editing, CRISPR/Cas9, BRCA1, DBR1, exon 18, single nucleotide variants, nonsense-mediated mRNA decay, exonic splicing enhancers, mRNA levels, cell fitness, variants of uncertain significance, cis-regulatory elements, trans-acting factors
- MeSH terms
- Cell Line, Exons, Humans, Regulatory Sequences, Nucleic Acid, RNA Nucleotidyltransferases, RNA Splicing, Templates, Genetic, Mutagenesis, Open Reading Frames, Conserved Sequence, Point Mutation, Genes, BRCA1, Genes, Essential, Genomics, Molecular Sequence Annotation, Nonsense Mediated mRNA Decay, Recombinational DNA Repair, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins
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