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      Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors

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          Abstract

          Base editing requires that the target sequence satisfy the PAM requirement of the Cas9 domain and that the target nucleotide is located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG PAMs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4–5 nucleotides to up to ~8–9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.

          Ed sum:

          Wider editing windows and different PAM requirements enable a broad set of genomic positions to be targeted with A and C base editors.

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          Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

          Kärt Tomberg, Michael Kosicki, Allan Bradley (2018)
          CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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            Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions

            Y Bill Kim, Alexis Komor, Jonathan Levy … (2017)
            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.
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              Precise base editing in rice, wheat and maize with a Cas9- cytidine deaminase fusion

              Yuan Zong, Yanpeng Wang, Chao Li … (2017)
              Single DNA base pairs are edited in wheat, rice and maize using a Cas9 nickase fusion protein.
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 9604648
                Journal ID (pubmed-jr-id): 20305
                Journal ID (nlm-ta): Nat Biotechnol
                Journal ID (iso-abbrev): Nat. Biotechnol.
                Title: Nature biotechnology
                ISSN (Print): 1087-0156
                ISSN (Electronic): 1546-1696
                Publication date Nihms-submitted: 12 April 2019
                Publication date (Electronic): 20 May 2019
                Publication date (Print): June 2019
                Publication date PMC-release: 20 November 2019
                Volume: 37
                Issue: 6
                Pages: 626-631
                Affiliations
                [1 ]Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
                [2 ]Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, 02138, USA
                [3 ]Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
                [4 ]Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 94720, USA
                [5 ]Innovative Genomics Institute, University of California, Berkeley, Berkeley CA, 94720, USA
                Author notes
                Correspondence should be addressed to David R. Liu: drliu@ 123456fas.harvard.edu

                Author contributions

                T.P.H. and K.T.Z. conducted the experiments, performed analyses, and wrote the manuscript. S.M.M. and N.M.G. conducted the experiments and performed analyses. B.L.O., C.F., and D.F.S. provided materials and advice on the CP variants. D.R.L. supervised the research and wrote the manuscript. All authors edited the manuscript.

                [*]

                These authors contributed equally to this work

                Article
                Manuscript ID: NIHMS1526897
                DOI: 10.1038/s41587-019-0134-y
                PMC ID: 6551276
                PubMed ID: 31110355
                SO-VID: 48bd4b68-c0fb-4c2c-befb-ecc8ead92730
                License:

                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

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                ScienceOpen disciplines: Biotechnology

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