A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

Plant Methods
Aftab A KhanGul Shad Ali

Abstract

Most current methods for constructing guide RNAs (gRNA) for the CRISPR/Cas9 genome editing system, depend on traditional cloning using specific type IIS restriction enzymes and DNA ligation. These methods consist of multiple steps of cloning, and are time consuming, resource intensive and not flexible. These issues are particularly exacerbated when multiple guide RNAs need to be assembled in one plasmid such as for multiplexing or for the paired nickases approach. Furthermore, identification of functional gRNA clones usually requires expensive in vitro screening. Addressing these issues will greatly facilitate usage and accessibility of CRISPR/Cas9 genome editing system to resource-limited laboratories. To improve efficiency of cloning multiple guide RNAs for the CRISPR/Cas9 system, we developed a restriction enzyme- and ligation-independent strategy for cloning gRNAs directly in plant expression vectors in one step. Our method relies on a negative selection marker and seamless cloning for combining multiple gRNAs directly in a plant expression vector in one reaction. In addition, using the Agrobacterium-mediated transient assays, this method provides a simple in planta procedure for assaying the effectiveness of multiple gRNAs...Continue Reading

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Citations

Jul 8, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ashraf S A El-SayedBasel Sitohy
Oct 18, 2020·Plants·Bed Prakash Bhatta, Subas Malla

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Methods Mentioned

BETA
PCR
transgenic

Software Mentioned

DNA

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