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Methods Protoc. 2018, 1(3), 29; https://doi.org/10.3390/mps1030029

High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates

1
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DU, UK
2
MRC WIMM Center for Computational Biology, Weatherall Institute of Molecular Medicine, Oxford OX3 9DU, UK
3
Genomics plc, Oxford OX1 1JD, UK
4
Weatherall Institute of Molecular Medicine, Oxford OX3 9DU, UK
5
Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, UK
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 25 June 2018 / Revised: 20 July 2018 / Accepted: 23 July 2018 / Published: 1 August 2018
(This article belongs to the Collection Gene Editing)
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Abstract

The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis. View Full-Text
Keywords: CRISPR/Cas9 genotyping; homology dependent repair; genome editing; genome-wide association study (GWAS) validation CRISPR/Cas9 genotyping; homology dependent repair; genome editing; genome-wide association study (GWAS) validation
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Nussbaum, L.; Telenius, J.M.; Hill, S.; Hirschfeld, P.P.; Suciu, M.C.; The WIGWAM Consortium; Downes, D.J.; Hughes, J.R. High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates. Methods Protoc. 2018, 1, 29.

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