Saturation editing of genomic regions by multiplex homology-directed repair

Abstract

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-harbor” 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 (HDR) 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.

Variant Interpretation: Functional Assays to the Rescue

“…In another example, saturation genome editing in a haploid human cell line was used for measuring the effects of most SNVs in a portion of the essential gene DBR1 (MIM: 607024). 15 In addition to complementation assays, cell-based protein-protein interaction assays could be applied in a general fashion to probe the effects of variants on disease-related interactions. This approach is bolstered by the fact that surveys of protein-protein interactions have revealed many interactions that, when disrupted, are deleterious.…”

“…46 Splicing and other RNAprocessing steps such as alternative polyadenylation are particularly well suited for investigation through MAVEs because distinct isoforms can be counted directly with RNA sequencing. MAVEs for splicing have already been implemented for both direct genome editing 15 and minigene assays. 14,47,48 MAVEs have also been developed to probe the effects of variants in untranslated regions of mRNAs on message stability and protein expression.…”

“…For example, genome-editing technologies are evolving, enabling the variants to be made and tested in their endogenous genomic context. 15,61,62 To capture cellular context, MAVEs can be performed in a cell type that is as close as possible to the diseaserelevant one. A more general solution to the context problem might be to collect MAVE data for each functional element in a panel of different cell types and assays.…”

“…For example, massively parallel reporter assays (MPRAs) query the effects of regulatory DNA variants on the expression of reporter genes, 13 whereas splicing assays reveal variant effects on mRNA processing. 14,15 The effect of variants on mRNA stability and translation can also be measured by multiplex assays. [16][17][18] Finally, deep mutational scans query the effect of amino acid substitutions on protein function.…”

Genome editing: A breakthrough in life science and medicine [Review]

“…Another example is the use of CRISPR/Cas9 for saturation mutagenesis, which is the generation of all possible mutations at a specific site or within narrow region of a gene; this is a powerful tool for both functional analysis of genes and directed evolution of functional enzymes. Saturation mutagenesis of an exon of an endogenous gene can be achieved by CRISPR/Cas9-guided cleavage of specific loci and multiplex homology-directed repair using a complex library of donor templates [31].…”