Publication Date:
2014-08-21
Description:
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.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156553/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156553/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Findlay, Gregory M -- Boyle, Evan A -- Hause, Ronald J -- Klein, Jason C -- Shendure, Jay -- DP1 HG007811/HG/NHGRI NIH HHS/ -- DP1HG007811/DP/NCCDPHP CDC HHS/ -- T32 GM007266/GM/NIGMS NIH HHS/ -- England -- Nature. 2014 Sep 4;513(7516):120-3. doi: 10.1038/nature13695.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA [2]. ; Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25141179" target="_blank"〉PubMed〈/a〉
Keywords:
CRISPR-Associated Proteins/metabolism
;
CRISPR-Cas Systems/genetics
;
Cell Line
;
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
;
Conserved Sequence/genetics
;
Exons/genetics
;
Genes, BRCA1
;
Genes, Essential/genetics
;
Genomics/*methods
;
Humans
;
Molecular Sequence Annotation/*methods
;
Mutagenesis/*genetics
;
Nonsense Mediated mRNA Decay
;
Open Reading Frames/genetics
;
Point Mutation/genetics
;
RNA Nucleotidyltransferases/genetics
;
RNA Splicing/genetics
;
Recombinational DNA Repair/*genetics
;
Regulatory Sequences, Nucleic Acid/genetics
;
Templates, Genetic
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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