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      Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I

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          Abstract

          Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (γPNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the γPNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA- Guided T7EI (PG-T7EI) technology demonstrates that T7EI can be used as a programmable nuclease capable of generating single or multiple specific DSBs in vitro under a broad range of conditions and could be potentially applied for large-scale genomic manipulation. With no protospacer adjacent motif (PAM) constraints and featuring a compact protein size, our PG-T7EI system will facilitate and expand DNA manipulations both in vitro and in vivo, including cloning, large-fragment DNA assembly, and gene editing, with exciting applications in biotechnology, medicine, agriculture, and synthetic biology.

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

          Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)
          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

            Alexis Komor, Yongjoo Kim, Michael Packer … (2016)
            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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              Search-and-replace genome editing without double-strand breaks or donor DNA

              Andrew Anzalone, Peyton B. Randolph, Jessie Davis … (2019)
              Summary Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 2–5 . 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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                Author and article information

                Contributors
                Rashid Aman:
                ORCID: https://orcid.org/0000-0002-6549-4887
                Muntjeeb M Syed
                Ahmed Saleh
                Firdaws Melliti
                Sivakrishna Rao Gundra
                Qiaochu Wang
                Tin Marsic
                Ahmed Mahas
                Magdy M Mahfouz:
                ORCID: https://orcid.org/0000-0002-0616-6365
                Journal
                Journal ID (nlm-ta): Nucleic Acids Res
                Journal ID (iso-abbrev): Nucleic Acids Res
                Journal ID (publisher-id): nar
                Title: Nucleic Acids Research
                Publisher: Oxford University Press
                ISSN (Print): 0305-1048
                ISSN (Electronic): 1362-4962
                Publication date Collection: 12 April 2024
                Publication date (Electronic): 29 February 2024
                Publication date PMC-release: 29 February 2024
                Volume: 52
                Issue: 6
                Pages: 3469-3482
                Affiliations
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Department of Genetics, Harvard University , Boston, MA 02115, USA
                Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology , Thuwal 23955-6900, Saudi Arabia
                Author notes
                To whom correspondence should be addressed. Tel: +966 2 808 2761; Email: magdy.mahfouz@ 123456kaust.edu.sa
                Author information
                https://orcid.org/0000-0002-6549-4887
                https://orcid.org/0000-0002-0616-6365
                Article
                Publisher ID: gkae148
                DOI: 10.1093/nar/gkae148
                PMC ID: 11014363
                PubMed ID: 38421613
                SO-VID: 8240a8c2-9bda-48f5-94e9-72c0493725cd
                Copyright © © The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

                History
                Date accepted : 18 February 2024
                Date revision received : 14 February 2024
                Date received : 01 November 2023
                Page count
                Pages: 14
                Funding
                Funded by: baseline;
                Award ID: BAS/1/1035-01-01
                Funded by: King Abdullah University of Science and Technology, DOI 10.13039/501100004052;
                Award ID: SHI/1/5490-01-01
                Categories
                Subject: AcademicSubjects/SCI00010
                Subject: Narese/29
                Subject: Synthetic Biology and Bioengineering

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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