Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing

Jiaying Yu, Xi Xiang, Jinrong Huang, Xue Liang, Xiaoguang Pan, Zhanying Dong, Trine Skov Petersen, Kunli Qu, Ling Yang, Xiaoying Zhao, Siyuan Li, Tianyu Zheng, Zhe Xu, Chengxun Liu, Peng Han, Fengping Xu, Huanming Yang, Xin Liu, Xiuqing Zhang, Lars BolundYonglun Luo, Lin Lin

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Abstract

Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.

OriginalsprogEngelsk
Artikelnummere25
TidsskriftNucleic Acids Research
Vol/bind48
Nummer5
Antal sider13
ISSN0305-1048
DOI
StatusUdgivet - 2020

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