High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates
AbstractThe 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
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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.
Nussbaum L, Telenius JM, Hill S, Hirschfeld PP, Suciu MC, The WIGWAM Consortium, Downes DJ, Hughes JR. High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates. Methods and Protocols. 2018; 1(3):29.Chicago/Turabian Style
Nussbaum, Lea; Telenius, Jelena M.; Hill, Stephanie; Hirschfeld, Priscila P.; Suciu, Maria C.; The WIGWAM Consortium; Downes, Damien J.; Hughes, Jim R. 2018. "High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates." Methods Protoc. 1, no. 3: 29.
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