Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems
- PMID: 27492474
- DOI: 10.1126/science.aaf8729
Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems
Abstract
The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.
Copyright © 2016, American Association for the Advancement of Science.
Comment in
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Harnessing mutation: The best of two worlds.Science. 2016 Sep 16;353(6305):1206-7. doi: 10.1126/science.aai8233. Science. 2016. PMID: 27634511 No abstract available.
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