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

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.

本数据集通过对HLA阴性的人类B细胞系721.221进行连续转导，成功构建了TAP缺陷型单等位基因HLA I类表达细胞系：所用慢病毒表达载体共分为三类，(i) 携带通过自剪切P2A肽（self-cleaving P2A peptide）连接的杀稻瘟菌素抗性基因的Cas9蛋白表达载体；(ii) 搭载18种不同HLA I类等位基因的表达载体，该组等位基因可实现超过99%的全球人群覆盖，具体包括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及Cw*0701，该载体通过内部核糖体进入序列（internal ribosome entry sequence, IRES）连接嘌呤霉素抗性基因以用于筛选；(iii) 靶向人类TAP1基因的单引导RNA（single guide RNA, sgRNA）表达载体，该载体通过IRES连接新霉素/G418抗性基因以用于筛选。随后，研究人员对每一株单等位基因细胞系的基因组DNA进行扩增子测序，验证了CRISPR/Cas9对TAP1基因的成功编辑。本次扩增子测序工作由麻省总医院（MGH）DNA测序核心实验室完成。