Design of Multiplexing CRISPR/Cas9 Constructs for Plant Genome Engineering Using the GoldenBraid DNA Assembly...

Area of Science:

• Plant Synthetic Biology
• Genome Engineering
• Molecular Cloning

Background:

• CRISPR/Cas9 technology offers significant potential for plant synthetic biology and genome engineering.
• Multiplexing guide RNAs (gRNAs) allows simultaneous targeting of multiple genomic loci, but construct design can be challenging for new users.

Purpose of the Study:

• To provide a straightforward method for creating multiplex CRISPR/Cas9 DNA constructs.
• To demonstrate the use of the GoldenBraid (GB) DNA assembly system for this purpose.
• To enable efficient simultaneous targeting of multiple genomic sites in plants.

Main Methods:

• Utilized the GoldenBraid (GB) DNA assembly system for constructing multiplex CRISPR/Cas9 vectors.
• Designed a polycistronic gRNA construct targeting three sites within the nopaline synthase promoter.
• Employed a dead Cas9 (dCas9) for transcriptional repression and validated through transient expression in Nicotiana benthamiana.

Main Results:

• Successfully created multiplex CRISPR/Cas9 constructs using the GoldenBraid system.
• Demonstrated transcriptional repression at three targeted loci via polycistronic gRNA expression.
• Validated the protocol's efficacy through transient expression assays with a luciferase reporter.

Conclusions:

• The GoldenBraid system offers an accessible and efficient method for generating multiplex CRISPR/Cas9 constructs in plants.
• This protocol facilitates advanced genome engineering strategies by enabling simultaneous modification of multiple genomic loci.
• The developed method supports applications in synthetic biology and functional genomics research.