figshare
Browse

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

Posted on 2017-10-16 - 05:00
Abstract Background 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. Results 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 very rapidly. Conclusions For a fraction of resources used in the type IIS restriction enzyme-based cloning method and in vitro screening assays, the system reported here allows efficient construction and testing several ready-to-transfect gRNA constructs in < 3 days. In addition, this system is highly versatile and flexible, and by designing only two additional target-specific primers, multiple gRNAs can be easily assembled in any plasmid in a single reaction.

CITE THIS COLLECTION

DataCite
3 Biotech
3D Printing in Medicine
3D Research
3D-Printed Materials and Systems
4OR
AAPG Bulletin
AAPS Open
AAPS PharmSciTech
Abhandlungen aus dem Mathematischen Seminar der Universität Hamburg
ABI Technik (German)
Academic Medicine
Academic Pediatrics
Academic Psychiatry
Academic Questions
Academy of Management Discoveries
Academy of Management Journal
Academy of Management Learning and Education
Academy of Management Perspectives
Academy of Management Proceedings
Academy of Management Review
or
Select your citation style and then place your mouse over the citation text to select it.

SHARE

email
need help?