Saturation mutagenesis1,2—coupled to an appropriate biological assay—represents a fundamental means of achieving a high-resolution understanding of regulatory3 and protein-coding4 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 locus5. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage6 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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Sequence Read Archive
Sequence data used for this analysis are available in SRA under accession number SRP044126.
We thank F. Zhang and his laboratory for the CRISPR/Cas9 backbone constructs used in this study and G. Church and his laboratory for providing reagents used to establish CRISPR/Cas9 editing techniques in our lab. We also thank members of the Shendure laboratory for helpful discussions and D. Prunkard for assistance with FACS. This work was supported by the National Institutes of Health (DP1HG007811 to J.S.) and the UW Medical Scientist Training Program (G.M.F. and J.K.).
Extended data figures
This table contains a list of oligonucleotide sequences used in this study.
This table contains enrichment scores from the BRCA1 exon 18 hexamer experiment.
This table contains effect sizes from the BRCA1 whole exon 18 SNV experiment.
This table contains enrichment scores from the DBR1 experiment.
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