Repositorium

What is a repositorium?

The repositorium is a searchable database that provides data on relevant articles from journals, company web pages and web pages of governmental agencies about studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Search options are article type, technique, plant, traits or free text. The repositorium is based on the systematic map of Dominik Modrzejewski et al., published in the journal environmental evidence. (Download article PDF).

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


Typ / Jahr

Journal Article / 2017

Autoren

Khan, Aftab A.; El-Sayed, Ashraf; Akbar, Asma; Mangravita-Novo, Arianna; Bibi, Shaheen; Afzal, Zunaira; Norman, David J.; Ali, Gul Shad

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.

Keywords
Periodical
Plant methods
Periodical Number
Page range
86
Volume
13
DOI
10.1186/s13007-017-0236-9

Techniques

ID Corresponding Author
Country
Plant Species GE Technique
Sequence Identifier
Trait
Type of Alteration
Progress in Research
Key Topic
1009 Ali, Gul Shad
USA
Nicotiana benthamiana CRISPR/Cas9
PDS
Albino phenotype
SDN1
Basic research
Basic research
1010 Ali, Gul Shad
USA
Nicotiana benthamiana CRISPR/Cas9
PIR2-1-mCherry
No information
SDN1
Basic research
Basic research
1011 Ali, Gul Shad
USA
Nicotiana benthamiana CRISPR/Cas9
YFP
No information
SDN1
Basic research
Basic research