Forskning ved Københavns Universitet - Københavns Universitet

Forside

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

Publikation: Bidrag til tidsskriftTidsskriftartikel

  • Jiaying Yu
  • Xi Xiang
  • 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 Bolund
  • Yonglun Luo
  • Lin Lin

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
Udgave nummer5
Antal sider13
ISSN0305-1048
DOI
StatusUdgivet - 2020

ID: 237997037