108
views
0
recommends
+1 Recommend
0 collections
    1
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Summary

          The spontaneous deamination of cytosine is a major source of C•G to T•A transitions, which account for half of known human pathogenic point mutations. The ability to efficiently convert target A•T base pairs to G•C therefore could advance the study and treatment of genetic diseases. While the deamination of adenine yields inosine, which is treated as guanine by polymerases, no enzymes are known to deaminate adenine in DNA. Here we report adenine base editors (ABEs) that mediate conversion of A•T to G•C in genomic DNA. We evolved a tRNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs ( e.g., ABE7.10), that convert target A•T to G•C base pairs efficiently (~50% in human cells) with very high product purity (typically ≥ 99.9%) and very low rates of indels (typically ≤ 0.1%). ABEs introduce point mutations more efficiently and cleanly than a current Cas9 nuclease-based method, induce less off-target genome modification than Cas9, and can install disease-correcting or disease-suppressing mutations in human cells. Together with our previous base editors, ABEs advance genome editing by enabling the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

          Related collections

          Most cited references34

          • Record: found
          • Abstract: found
          • Article: not found

          Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions

          Base editing is a recently developed approach to genome editing that uses a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single-nucleotide changes in the DNA of living cells without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions 1 . Here we report the development of five new C→T (or G→A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing by 2.5-fold. Additionally, we engineered new base editors containing mutated cytidine deaminase domains that narrow the width of the apparent editing window from approximately 5 nucleotides to as little as 1 to 2 nucleotides, enabling the discrimination of neighboring C nucleotides that would previously be edited with comparable efficiency, thereby doubling the number of disease-associated target Cs that can be corrected preferentially over nearby non-target Cs. Collectively, these developments substantially increase the targeting scope of base editing and establish the modular nature of base editors.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Precise base editing in rice, wheat and maize with a Cas9- cytidine deaminase fusion

            Single DNA base pairs are edited in wheat, rice and maize using a Cas9 nickase fusion protein.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Highly efficient RNA-guided base editing in mouse embryos

              Mice with targeted point mutations are generated efficiently using Cas9–cytidine deaminase fusions.
                Bookmark

                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                18 October 2017
                25 October 2017
                23 November 2017
                25 April 2018
                : 551
                : 7681
                : 464-471
                Affiliations
                [1 ]Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, 02138
                [2 ]Howard Hughes Medical Institute, Harvard University, Cambridge, MA, 02138
                [3 ]Broad Institute of MIT and Harvard, Cambridge, MA, 02141
                Author notes
                [* ]Correspondence should be addressed to David R. Liu: drliu@ 123456fas.harvard.edu
                Article
                NIHMS913981
                10.1038/nature24644
                5726555
                29160308
                e5a6663f-a378-454b-b5d8-7279497dc446

                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Reprints and permissions information is available at www.nature.com/reprints.

                History
                Categories
                Article

                Uncategorized
                Uncategorized

                Comments

                Comment on this article