CRISPR amplicon sequencing of human TAP1 gene of CRISPR/Cas9-edited mono-allelic HLA class I-expressing cell lines

Published: 29 October 2020| Version 1 | DOI: 10.17632/hh46ymyxkk.1
Contributor:
Anusha Nathan

Description

TAP-deficient monoallelic HLA class I-expressing cell lines were generated by sequential transduction of the HLA-null human B cell line 721.221 with lentiviral expression constructs encoding (i) Cas9 protein linked to blasticidin resistance gene via a self-cleaving P2A peptide, (ii) 18 distinct HLA class I alleles that provided >99% global coverage (A*0101, A*0201, A*0301, A*2402, B*0702, B*0801, B*1402, B*1501, B*2705, B*3501, B*3901, B*4001, B*4402, B*5201, B*5701, B*5801, B*8101 and Cw*0701) under puromycin resistance linked via an internal ribosome entry sequence (IRES), and (iii) a single guide RNA (sgRNA) directed towards human TAP1 under neomycin/G418 resistance linked via an IRES. Successful CRISPR/Cas9 editing of the TAP1 gene was confirmed by amplicon sequencing of genomic DNA for each mono-allelic cell line. Amplicon sequencing was carried out by the MGH DNA Sequencing Core Facility.

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