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Pitfalls in Single Clone CRISPR-Cas9 Mutagenesis to Fine-Map Regulatory Intervals

Center for Integrative Genomics, Georgia Institute of Technology, Atlanta, GA 30332, USA
Systems, Synthetic, and Physical Biology, Rice University, Houston, TX 77005, USA
Department of Bioengineering, Rice University, Houston, TX 77005, USA
APC Microbiome Ireland, University College Cork, Cork T12 YN60, Ireland
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Current Affiliation: School of Medicine, University of Texas Health Science Center at San Antonio, TX 78229, USA.
Genes 2020, 11(5), 504;
Received: 19 March 2020 / Revised: 15 April 2020 / Accepted: 22 April 2020 / Published: 4 May 2020
The 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. View Full-Text
Keywords: eQTL; CRISPR-Cas9; single-cell clone; fine-mapping; power eQTL; CRISPR-Cas9; single-cell clone; fine-mapping; power
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MDPI and ACS Style

Tian, R.; Pan, Y.; Etheridge, T.H.A.; Deshmukh, H.; Gulick, D.; Gibson, G.; Bao, G.; Lee, C.M. Pitfalls in Single Clone CRISPR-Cas9 Mutagenesis to Fine-Map Regulatory Intervals. Genes 2020, 11, 504.

AMA Style

Tian R, Pan Y, Etheridge THA, Deshmukh H, Gulick D, Gibson G, Bao G, Lee CM. Pitfalls in Single Clone CRISPR-Cas9 Mutagenesis to Fine-Map Regulatory Intervals. Genes. 2020; 11(5):504.

Chicago/Turabian Style

Tian, Ruoyu, Yidan Pan, Thomas H.A. Etheridge, Harshavardhan Deshmukh, Dalia Gulick, Greg Gibson, Gang Bao, and Ciaran M. Lee. 2020. "Pitfalls in Single Clone CRISPR-Cas9 Mutagenesis to Fine-Map Regulatory Intervals" Genes 11, no. 5: 504.

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