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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.
Price: $32.50
Publisher: Burleigh Dodds Science Publishing
Imprint: Burleigh Dodds Science Publishing
Series: Burleigh Dodds Series in Agricultural Science
Publication Date:
19 April 2021
ISBN: 9781801461399
Format: eBook
BISACs:
TECHNOLOGY & ENGINEERING / Agriculture / Agronomy / Crop Science, TECHNOLOGY & ENGINEERING / Agriculture / Sustainable Agriculture
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