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      Cas9 degradation in human cells using phage anti-CRISPR proteins

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          Abstract

          Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here, we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. AcrIIA1-induced degradation of SpyCas9 is abolished by mutations in AcrIIA1 that break a direct physical interaction between the 2 proteins. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.

          Abstract

          Bacteriophages encode anti-CRISPR proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. This study shows that the anti-CRISPR protein AcrIIA1 induces degradation of Cas9 in human cells.

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          Most cited references24

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

            Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. 1,2 Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus from the cellular response to dsDNA breaks. 1,2 Here we report the development of base editing, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides (nt), and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of ∼15-75% of total cellular DNA with minimal (typically ≤ 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
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              CRISPR provides acquired resistance against viruses in prokaryotes.

              Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.
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                Author and article information

                Contributors
                Role: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Writing – original draft
                Role: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Writing – review & editing
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                PLOS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                8 December 2023
                December 2023
                8 December 2023
                : 21
                : 12
                : e3002431
                Affiliations
                [1 ] Acrigen Biosciences, Inc., Alameda, California, United States of America
                [2 ] Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, California, United States of America
                University of Southampton, UNITED KINGDOM
                Author notes

                D.R. and J.B.D. are founders of Acrigen Biosciences. D.R., M.S., Z.M., and L.T. are employees of Acrigen Biosciences.

                Author information
                https://orcid.org/0000-0002-9494-5464
                Article
                PBIOLOGY-D-23-00767
                10.1371/journal.pbio.3002431
                10732428
                38064533
                a5547d76-25e0-4c09-8aad-3a01ce653023
                © 2023 Meacham et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 23 March 2023
                : 14 November 2023
                Page count
                Figures: 3, Tables: 0, Pages: 9
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: R43GM145002
                Award Recipient :
                This work was supported by the National Institute of General Medical Sciences (R43GM145002) to DR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Discovery Report
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Precipitation Techniques
                Immunoprecipitation
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Nucleases
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Hydrolases
                Nucleases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Hydrolases
                Nucleases
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Research and analysis methods
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Biology and Life Sciences
                Organisms
                Viruses
                Bacteriophages
                Biology and Life Sciences
                Organisms
                Bacteria
                Listeria
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Biology and Life Sciences
                Immunology
                Immune System Proteins
                Medicine and Health Sciences
                Immunology
                Immune System Proteins
                Biology and Life Sciences
                Biochemistry
                Proteins
                Immune System Proteins
                Custom metadata
                vor-update-to-uncorrected-proof
                2023-12-20
                All relevant data are within the paper and its Supporting Information files.

                Life sciences
                Life sciences

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