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      Adenine base editors catalyze cytosine conversions in human cells

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

          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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            Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

            Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C->T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.
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              Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing

              A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.
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                Author and article information

                Journal
                Nature Biotechnology
                Nat Biotechnol
                Springer Science and Business Media LLC
                1087-0156
                1546-1696
                September 23 2019
                Article
                10.1038/s41587-019-0254-4
                © 2019

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