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      Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity

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          Abstract

          Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), resulting in ABE8e. ABE8e contains eight additional mutations that increase activity (k app) 590-fold compared with ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disrupting regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by introducing additional mutations in the TadA-8e domain. Finally, we show that ABE8e can efficiently edit natural mutations in a GATA1 binding site in the BCL11A enhancer or the HBG promoter in human cells, targets which were poorly edited with ABE7.10. ABE8e broadens the effectiveness and applicability of adenine base editing.

          Editorial Summary

          A continuously evolved adenine base editor is compatible with various Cas proteins and mediates efficient A•T-to-G•C base conversions at a wide variety of PAM sites.

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          Most cited references 31

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          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.
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            BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

            Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements.
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              Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery

              We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo.
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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                1087-0156
                1546-1696
                12 June 2020
                16 March 2020
                July 2020
                16 September 2020
                : 38
                : 7
                : 883-891
                Affiliations
                [1 ]Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
                [2 ]Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
                [3 ]Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
                [4 ]Department of Molecular and Cell Biology, University of California, Berkeley, CA.
                [5 ]Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA.
                [6 ]Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA.
                [7 ]Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
                [8 ]Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA.
                [9 ]Howard Hughes Medical Institute, University of California, Berkeley, CA.
                [10 ]Innovative Genomics Institute, University of California, Berkeley, CA.
                [11 ]Department of Chemistry, University of California, Berkeley, CA.
                [12 ]Present address: School of Molecular Sciences, Arizona State University, Tempe, AZ.
                [13 ]Present address: Department of Chemistry, Williams College, Williamstown, MA.
                Author notes
                [*]

                These authors contributed equally: M. F. Richter, K. T. Zhao.

                Author contributions

                M.F.R. and K.T.Z. conducted the experiments, performed analyses, and wrote the manuscript. E.E., G.A.N, A.L., B.W.T, C.W., and L.W.K. conducted the experiments and performed analyses. J.Z. and D.E.B. provided information on disease loci. J.A.D. provided feedback on biochemical analyses. D.R.L supervised the research and wrote the manuscript. All authors edited the manuscript.

                Correspondence should be addressed to David R. Liu: drliu@ 123456fas.harvard.edu
                Article
                NIHMS1558603
                10.1038/s41587-020-0453-z
                7357821
                32433547

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                Biotechnology

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