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      Search-and-replace genome editing without double-strand breaks or donor DNA

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          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

          Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 25 . Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various tags and epitopes into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct up to 89% of known genetic variants associated with human diseases.

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          Most cited references16

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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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            CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.

            Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.
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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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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                19 October 2019
                21 October 2019
                December 2019
                21 April 2020
                : 576
                : 7785
                : 149-157
                Affiliations
                [1 ]Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA
                [2 ]Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA
                [3 ]Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA
                Author notes
                [* ]Correspondence should be addressed to David R. Liu: drliu@ 123456fas.harvard.edu

                Author contributions

                A.V.A. designed the research, performed experiments, analyzed data, and wrote the manuscript. P.B.R., J.R.D., A.A.S., and G.A.N. performed human cell experiments and analyzed data. L.W.K. and J.M.L. performed neuron experiments. P.J.C. and C.W. performed and analyzed RNA-seq experiments. A.R. analyzed ClinVar. D.R.L designed and supervised the research and wrote the manuscript.

                Article
                NIHMS1541141
                10.1038/s41586-019-1711-4
                6907074
                31634902
                8ad218f2-b7c3-4fad-aa82-24b4a3ac6b0c

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