| RRC ID |
62315
|
| 著者 |
Nussbaum L, Telenius JM, Hill S, Hirschfeld PP, Suciu MC, WIGWAM Consortium, Downes DJ, Hughes JR.
|
| タイトル |
High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates.
|
| ジャーナル |
Methods Protoc
|
| 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.
|
| 巻・号 |
1(3)
|
| 公開日 |
2018-8-1
|
| DOI |
10.3390/mps1030029
|
| PII |
mps1030029
|
| PMID |
31164571
|
| PMC |
PMC6481090
|
| リソース情報 |
| ヒト・動物細胞 |
HUDEP-2(RCB4557) |
