Pitfalls in single clone crispr-cas9 mutagenesis to fine-map regulatory intervals

dc.contributor.authorTian, Ruoyu
dc.contributor.authorPan, Yidan
dc.contributor.authorEtheridge, Thomas H. A.
dc.contributor.authorDeshmukh, Harshavardhan
dc.contributor.authorGulick, Dalia
dc.contributor.authorGibson, Greg
dc.contributor.authorBao, Gang
dc.contributor.authorLee, Ciaran M.
dc.contributor.funderNational Institutes of Healthen
dc.date.accessioned2021-01-15T11:03:22Z
dc.date.available2021-01-15T11:03:22Z
dc.date.issued2020-05-04
dc.date.updated2021-01-15T10:49:25Z
dc.description.abstractThe majority of genetic variants affecting complex traits map to regulatory regions of genes, and typically lie in credible intervals of 100 or more SNPs. Fine mapping of the causal variant(s) at a locus depends on assays that are able to discriminate the effects of polymorphisms or mutations on gene expression. Here, we evaluated a moderate-throughput CRISPR-Cas9 mutagenesis approach, based on replicated measurement of transcript abundance in single-cell clones, by deleting candidate regulatory SNPs, affecting four genes known to be affected by large-effect expression Quantitative Trait Loci (eQTL) in leukocytes, and using Fluidigm qRT-PCR to monitor gene expression in HL60 pro-myeloid human cells. We concluded that there were multiple constraints that rendered the approach generally infeasible for fine mapping. These included the non-targetability of many regulatory SNPs, clonal variability of single-cell derivatives, and expense. Power calculations based on the measured variance attributable to major sources of experimental error indicated that typical eQTL explaining 10% of the variation in expression of a gene would usually require at least eight biological replicates of each clone. Scanning across credible intervals with this approach is not recommended.en
dc.description.sponsorshipNational Institutes of Health (Project number: 1R01HG008146-01A)en
dc.description.statusPeer revieweden
dc.description.versionPublished Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.articleid504en
dc.identifier.citationTian, R., Pan, Y., Etheridge, T. H. A., Deshmukh, H., Gulick, D., Gibson, G., Bao, G. and Lee, C. M. (2020) 'Pitfalls in single clone crispr-cas9 mutagenesis to fine-map regulatory intervals', Genes, 11(5), 504 (18pp). doi: 10.3390/genes11050504en
dc.identifier.doi10.3390/genes11050504en
dc.identifier.endpage18en
dc.identifier.issn2073-4425
dc.identifier.issued5en
dc.identifier.journaltitleGenesen
dc.identifier.startpage1en
dc.identifier.urihttps://hdl.handle.net/10468/10921
dc.identifier.volume11en
dc.language.isoenen
dc.publisherMDPIen
dc.relation.ispartofSpecial Issue: Computational Methods for the Analysis of Genomic Data and Biological Processes
dc.rights© 2020, the Authors. Published under licence by MDPI. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.en
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en
dc.subjectCRISPR-Cas9en
dc.subjecteQTLen
dc.subjectFine-mappingen
dc.subjectPoweren
dc.subjectSingle-cell cloneen
dc.titlePitfalls in single clone crispr-cas9 mutagenesis to fine-map regulatory intervalsen
dc.typeArticle (peer-reviewed)en
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