Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing.
Nucleic Acids Res
; 48(5): e25, 2020 03 18.
Article
em En
| MEDLINE
| ID: mdl-31943080
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA Circular
/
RNA Guia de Cinetoplastídeos
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
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Sistemas CRISPR-Cas
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Proteína 9 Associada à CRISPR
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
China