Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair

By Gregory M. Findlay, Evan A. Boyle, Ronald J. Hause, Jason C. Klein, Jay Shendure. 2014

368 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

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Citations in the corpus

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, neutral): “Primers and templates used in the synthesis of all sgRNA plasmids used in this work are listed in Supplementary Tables 8, 9, 11, 12, 15, and 16.”
Learning the sequence determinants of alternative splicing from millions of random sequences. (2015, background): “In fact, recent work demonstrated that small-scale genomic libraries could be created through insertion of degenerate sequences directly into an alternatively spliced gene locus (Findlay et al., 2014).”
CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling (2014, background): “Given recent successes using Cas9 and large-scale sgRNA libraries for genetic screening (Findlay et al., 2014; Koike-Yusa et al., 2014; Sanjana et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014), the Cas9 mouse may be directly combined with genome-wide or targeted sgRNA…”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “…can edit DNA is changing the pace of biological research in many areas, including identifying and understanding mechanisms of genetic diseases (Findlay et al., 2014; Gilbert et al., 2014; Zhou et al., 2014; Konermann et al., 2015), validating disease targets (Shalem et al., 2014; Wang et al.,…”