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  1. Article ; Online: Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

    Bischoff, Nadja / Wimberger, Sandra / Maresca, Marcello / Brakebusch, Cord

    Cells

    2020  Volume 9, Issue 5

    Abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug ... ...

    Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.
    MeSH term(s) Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; DNA Damage/genetics ; DNA Repair/genetics ; Gene Editing ; Humans ; Small Molecule Libraries/metabolism
    Chemical Substances Small Molecule Libraries
    Language English
    Publishing date 2020-05-25
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 2661518-6
    ISSN 2073-4409 ; 2073-4409
    ISSN (online) 2073-4409
    ISSN 2073-4409
    DOI 10.3390/cells9051318
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing.

    Peterka, Martin / Akrap, Nina / Li, Songyuan / Wimberger, Sandra / Hsieh, Pei-Pei / Degtev, Dmitrii / Bestas, Burcu / Barr, Jack / van de Plassche, Stijn / Mendoza-Garcia, Patricia / Šviković, Saša / Sienski, Grzegorz / Firth, Mike / Maresca, Marcello

    Nature communications

    2022  Volume 13, Issue 1, Page(s) 1240

    Abstract: Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor ( ... ...

    Abstract Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.
    MeSH term(s) Animals ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; Endonucleases/metabolism ; Gene Editing ; Mammals/genetics
    Chemical Substances Endonucleases (EC 3.1.-)
    Language English
    Publishing date 2022-03-24
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-022-28771-1
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo.

    Bestas, Burcu / Wimberger, Sandra / Degtev, Dmitrii / Madsen, Alexandra / Rottner, Antje K / Karlsson, Fredrik / Naumenko, Sergey / Callahan, Megan / Touza, Julia Liz / Francescatto, Margherita / Möller, Carl Ivar / Badertscher, Lukas / Li, Songyuan / Cerboni, Silvia / Selfjord, Niklas / Ericson, Elke / Gordon, Euan / Firth, Mike / Chylinski, Krzysztof /
    Taheri-Ghahfarokhi, Amir / Bohlooly-Y, Mohammad / Snowden, Mike / Pangalos, Menelaos / Nuttall, Barrett / Akcakaya, Pinar / Sienski, Grzegorz / Maresca, Marcello

    Nature communications

    2023  Volume 14, Issue 1, Page(s) 5474

    Abstract: Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have ... ...

    Abstract Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.
    MeSH term(s) Animals ; Mice ; Translocation, Genetic ; Proprotein Convertase 9/genetics ; CRISPR-Cas Systems/genetics ; Mutation ; Endonucleases/genetics ; Streptococcus pyogenes/genetics
    Chemical Substances PCSK9 protein, human (EC 3.4.21.-) ; Proprotein Convertase 9 (EC 3.4.21.-) ; Endonucleases (EC 3.1.-)
    Language English
    Publishing date 2023-09-06
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-023-41240-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing.

    Wimberger, Sandra / Akrap, Nina / Firth, Mike / Brengdahl, Johan / Engberg, Susanna / Schwinn, Marie K / Slater, Michael R / Lundin, Anders / Hsieh, Pei-Pei / Li, Songyuan / Cerboni, Silvia / Sumner, Jonathan / Bestas, Burcu / Schiffthaler, Bastian / Magnusson, Björn / Di Castro, Silvio / Iyer, Preeti / Bohlooly-Y, Mohammad / Machleidt, Thomas /
    Rees, Steve / Engkvist, Ola / Norris, Tyrell / Cadogan, Elaine B / Forment, Josep V / Šviković, Saša / Akcakaya, Pinar / Taheri-Ghahfarokhi, Amir / Maresca, Marcello

    Nature communications

    2023  Volume 14, Issue 1, Page(s) 4761

    Abstract: Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations ... ...

    Abstract Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (Polϴ). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.
    MeSH term(s) Gene Editing ; CRISPR-Cas Systems/genetics ; Protein Kinases/genetics ; DNA Repair/genetics ; DNA/genetics
    Chemical Substances Protein Kinases (EC 2.7.-) ; DNA (9007-49-2)
    Language English
    Publishing date 2023-08-14
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-023-40344-4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: TCR Fingerprinting and Off-Target Peptide Identification.

    Karapetyan, Armen R / Chaipan, Chawaree / Winkelbach, Katharina / Wimberger, Sandra / Jeong, Jun Seop / Joshi, Bishnu / Stein, Robert B / Underwood, Dennis / Castle, John C / van Dijk, Marc / Seibert, Volker

    Frontiers in immunology

    2019  Volume 10, Page(s) 2501

    Abstract: Adoptive T cell therapy using patient T cells redirected to recognize tumor-specific antigens by expressing genetically engineered high-affinity T-cell receptors (TCRs) has therapeutic potential for melanoma and other solid tumors. Clinical trials ... ...

    Abstract Adoptive T cell therapy using patient T cells redirected to recognize tumor-specific antigens by expressing genetically engineered high-affinity T-cell receptors (TCRs) has therapeutic potential for melanoma and other solid tumors. Clinical trials implementing genetically modified TCRs in melanoma patients have raised concerns regarding off-target toxicities resulting in lethal destruction of healthy tissue, highlighting the urgency of assessing which off-target peptides can be recognized by a TCR. As a model system we used the clinically efficacious NY-ESO-1-specific TCR C
    MeSH term(s) Biological Assay ; Cell Line, Tumor ; Epitopes, T-Lymphocyte/analysis ; Epitopes, T-Lymphocyte/chemistry ; Epitopes, T-Lymphocyte/genetics ; Epitopes, T-Lymphocyte/immunology ; HEK293 Cells ; Humans ; Lymphocyte Activation ; Peptides/analysis ; Peptides/chemistry ; Peptides/genetics ; Peptides/immunology ; Receptors, Antigen/genetics ; Receptors, Antigen/immunology ; T-Lymphocytes/cytology ; T-Lymphocytes/immunology
    Chemical Substances Epitopes, T-Lymphocyte ; Peptides ; Receptors, Antigen
    Language English
    Publishing date 2019-10-22
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2606827-8
    ISSN 1664-3224 ; 1664-3224
    ISSN (online) 1664-3224
    ISSN 1664-3224
    DOI 10.3389/fimmu.2019.02501
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Author Correction: Universal toxin-based selection for precise genome engineering in human cells.

    Li, Songyuan / Akrap, Nina / Cerboni, Silvia / Porritt, Michelle J / Wimberger, Sandra / Lundin, Anders / Möller, Carl / Firth, Mike / Gordon, Euan / Lazovic, Bojana / Sieńska, Aleksandra / Pane, Luna Simona / Coelho, Matthew A / Ciotta, Giovanni / Pellegrini, Giovanni / Sini, Marcella / Xu, Xiufeng / Mitra, Suman / Bohlooly-Y, Mohammad /
    Taylor, Benjamin J M / Sienski, Grzegorz / Maresca, Marcello

    Nature communications

    2021  Volume 12, Issue 1, Page(s) 2832

    Language English
    Publishing date 2021-05-10
    Publishing country England
    Document type Published Erratum
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-021-23345-z
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Universal toxin-based selection for precise genome engineering in human cells.

    Li, Songyuan / Akrap, Nina / Cerboni, Silvia / Porritt, Michelle J / Wimberger, Sandra / Lundin, Anders / Möller, Carl / Firth, Mike / Gordon, Euan / Lazovic, Bojana / Sieńska, Aleksandra / Pane, Luna Simona / Coelho, Matthew A / Ciotta, Giovanni / Pellegrini, Giovanni / Sini, Marcella / Xu, Xiufeng / Mitra, Suman / Bohlooly-Y, Mohammad /
    Taylor, Benjamin J M / Sienski, Grzegorz / Maresca, Marcello

    Nature communications

    2021  Volume 12, Issue 1, Page(s) 497

    Abstract: Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based ... ...

    Abstract Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.
    MeSH term(s) Animals ; CD4-Positive T-Lymphocytes/cytology ; CD4-Positive T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Cell Proliferation/genetics ; Cell Survival/genetics ; Cells, Cultured ; Gene Editing/methods ; Genetic Engineering/methods ; HCT116 Cells ; HEK293 Cells ; Heparin-binding EGF-like Growth Factor/genetics ; Humans ; Induced Pluripotent Stem Cells/cytology ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Mutation
    Chemical Substances Heparin-binding EGF-like Growth Factor
    Language English
    Publishing date 2021-01-21
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-020-20810-z
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.

    Lundin, Anders / Porritt, Michelle J / Jaiswal, Himjyot / Seeliger, Frank / Johansson, Camilla / Bidar, Abdel Wahad / Badertscher, Lukas / Wimberger, Sandra / Davies, Emma J / Hardaker, Elizabeth / Martins, Carla P / James, Emily / Admyre, Therese / Taheri-Ghahfarokhi, Amir / Bradley, Jenna / Schantz, Anna / Alaeimahabadi, Babak / Clausen, Maryam / Xu, Xiufeng /
    Mayr, Lorenz M / Nitsch, Roberto / Bohlooly-Y, Mohammad / Barry, Simon T / Maresca, Marcello

    Nature communications

    2020  Volume 11, Issue 1, Page(s) 4903

    Abstract: The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with ...

    Abstract The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.
    MeSH term(s) Animals ; Antineoplastic Agents/pharmacology ; Antineoplastic Agents/therapeutic use ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/drug therapy ; Carcinoma, Non-Small-Cell Lung/genetics ; Cell Line, Tumor ; Doxycycline/pharmacology ; Drug Discovery/methods ; Drug Screening Assays, Antitumor/methods ; Female ; Gene Editing/methods ; Gene Expression/drug effects ; Gene Expression/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Genetic Vectors/genetics ; HEK293 Cells ; High-Throughput Screening Assays/methods ; Humans ; Lung Neoplasms/drug therapy ; Lung Neoplasms/genetics ; Male ; Mice ; Mice, Transgenic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Recombination, Genetic/drug effects ; Reproducibility of Results ; Transcriptional Activation/drug effects ; Transfection/methods ; Transgenes/genetics
    Chemical Substances Antineoplastic Agents ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Associated Protein 9 (EC 3.1.-) ; Cas9 endonuclease Streptococcus pyogenes (EC 3.1.-) ; Doxycycline (N12000U13O)
    Language English
    Publishing date 2020-09-29
    Publishing country England
    Document type Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-020-18548-9
    Database MEDical Literature Analysis and Retrieval System OnLINE

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