Institut for Biomedicin

Lars Bolund

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

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DOI

  • Jiaying Yu, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Xi Xiang
  • Jinrong Huang
  • Xue Liang, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Xiaoguang Pan, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Zhanying Dong, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Trine Skov Petersen
  • Kunli Qu, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Ling Yang, BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China.
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  • Xiaoying Zhao, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Siyuan Li, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Tianyu Zheng, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Zhe Xu, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Chengxun Liu, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Peng Han, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Fengping Xu, BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China.
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  • Huanming Yang, BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China.
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  • Xin Liu, BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China.
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  • Xiuqing Zhang, BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China.
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  • 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
Nummer5
Antal sider13
ISSN0305-1048
DOI
StatusUdgivet - 2020

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