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Advances in assembling gRNA/Cas9 constructs in genome editing of plants

Code: 9781801461399
Marta Vázquez Vilar, Sara Selma, Asun Fernández del Carmen and Diego Orzáez, Instituto de Biología Molecular and Celular de Plantas de Valencia (CSIC-UPV), Spain

Chapter synopsis: This chapter reviews the principles of CRISPR cloning in binary vectors and the different methods and elements employed, including the nucleases alternative to Cas9. It pays special attention to modular cloning strategies and multiplexing tools as well as the engineering of expanded Cas activities. Finally, the chapter includes a case study of the cloning of a nine gRNA multiplex construct and the analysis of its transformants in tobacco plants.

DOI: 10.19103/AS.2020.0082.10
Table of contents 1 Introduction 2 Modular cloning tools for assembling CRISPR/Cas constructs 3 The basics of CRISPR/Cas9 cloning 4 CRISPR-Cas12 specific cloning requirements 5 Expanding CRISPR/Cas9 activities with modular cloning 6 The polycistronic strategy for gRNA multiplexing 7 Case study: multiple gene targeting in tobacco with GoldenBraid cloning 8 References

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