Saturation Genome Editing by Multiplex Homologous Donor Repair. G. M. Findlay, E. A. Boyle, R. J. Hause, J. Klein, J. Shendure Department of Genome Sciences, University of Washington, Seattle, WA.

   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 interrogated by episomal expression or random genomic integration fail to account for the native context of the element's chromosomal locus with respect to the surrounding sequence, epigenetic milieu, and endogenous transcriptional activity. 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 to introduce hundreds to thousands of programmed variants within a single population of cells, a method we call saturation genome editing. Furthermore, we show that despite the relatively low fraction of edited cells, massively parallel functional analysis of such populations is possible by performing selective amplification of RNA or DNA from edited genomes followed by high-throughput sequencing. As a proof-of-concept, 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 substitutions, and measure strong and reproducible effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We next perform saturation genome editing in haploid human cells, targeting a well-conserved 75 bp coding region of an essential gene, DBR1. Measurement of mutations relative effects on growth span over three orders of magnitude for missense SNVs, correlate with predictive models of functional impact, and reveal only few conservative amino acid substitutions are well-tolerated in the enzymes active site. These proof-of-concept experiments show that saturation genome editing enables the multiplex functional analysis of mutations within the context of the genome itself. This approach facilitates the precise measurement of the consequences of large numbers of genomic mutations and may potentially aid in the interpretation of variants of uncertain significance observed in clinical sequencing.

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