Journal
METHODS AND PROTOCOLS
Volume 1, Issue 3, Pages -Publisher
MDPI
DOI: 10.3390/mps1030029
Keywords
CRISPR; Cas9 genotyping; homology dependent repair; genome editing; genome-wide association study (GWAS) validation
Categories
Funding
- Wellcome Trust strategic award [106130/Z/14/Z]
- Medical Research Council [MC_UU_12009/15]
- Wellcome Trust [106130/Z/14/Z] Funding Source: Wellcome Trust
- MRC [MC_UU_12009/15, MC_UU_00016/14] Funding Source: UKRI
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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.
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