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Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

Author

Listed:
  • Jeremy Vicencio

    (Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL)

  • Carlos Sánchez-Bolaños

    (Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • Ismael Moreno-Sánchez

    (Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • David Brena

    (Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL)

  • Charles E. Vejnar

    (Yale University School of Medicine)

  • Dmytro Kukhtar

    (Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL)

  • Miguel Ruiz-López

    (Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL)

  • Mariona Cots-Ponjoan

    (Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL)

  • Alejandro Rubio

    (Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • Natalia Rodrigo Melero

    (Biomolecular Screening and Protein Technologies Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology)

  • Jesús Crespo-Cuadrado

    (Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía)

  • Carlo Carolis

    (Biomolecular Screening and Protein Technologies Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology)

  • Antonio J. Pérez-Pulido

    (Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • Antonio J. Giráldez

    (Yale University School of Medicine
    Yale University School of Medicine
    Yale University School of Medicine)

  • Benjamin P. Kleinstiver

    (Center for Genomic Medicine, Massachusetts General Hospital
    Massachusetts General Hospital
    Harvard Medical School)

  • Julián Cerón

    (Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL)

  • Miguel A. Moreno-Mateos

    (Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

Abstract

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

Suggested Citation

  • Jeremy Vicencio & Carlos Sánchez-Bolaños & Ismael Moreno-Sánchez & David Brena & Charles E. Vejnar & Dmytro Kukhtar & Miguel Ruiz-López & Mariona Cots-Ponjoan & Alejandro Rubio & Natalia Rodrigo Meler, 2022. "Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-30228-4
    DOI: 10.1038/s41467-022-30228-4
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    References listed on IDEAS

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    1. Giedrius Gasiunas & Joshua K. Young & Tautvydas Karvelis & Darius Kazlauskas & Tomas Urbaitis & Monika Jasnauskaite & Mantvyda M. Grusyte & Sushmitha Paulraj & Po-Hao Wang & Zhenglin Hou & Shane K. Do, 2020. "A catalogue of biochemically diverse CRISPR-Cas9 orthologs," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
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    Cited by:

    1. Yu Zhang & Yang Liu & Wei Qin & Shaohui Zheng & Jiawang Xiao & Xinxin Xia & Xuanyao Yuan & Jingjing Zeng & Yu Shi & Yan Zhang & Hui Ma & Gaurav K. Varshney & Ji-Feng Fei & Yanmei Liu, 2024. "Cytosine base editors with increased PAM and deaminase motif flexibility for gene editing in zebrafish," Nature Communications, Nature, vol. 15(1), pages 1-10, December.
    2. Marion Rosello & Malo Serafini & Luca Mignani & Dario Finazzi & Carine Giovannangeli & Marina C. Mione & Jean-Paul Concordet & Filippo Del Bene, 2022. "Disease modeling by efficient genome editing using a near PAM-less base editor in vivo," Nature Communications, Nature, vol. 13(1), pages 1-13, December.

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