Comprehensive approaches to design efficient gRNA for SDN1-CRISPR/Cas9 genome editing in wheat

Tushadri Singh^1,2Mamrutha HM^2*Rajinder Singh^2*Jai Prakash Jaiswal¹Zeenat Wadhwa²Rakesh Kumar²Omvir Singh²Om Prakhash Ahlawat²Ratan Tiwari²

• ¹Department of Genetics and Plant Breeding, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India
• ²Indian Institute of Wheat and Barley Research (ICAR), Karnal, India

Background: CRISPR/Cas9 technology has gained popularity due to its efficient, widely applicable and relatively easy editing of genome. Further, the removal of regulation on Site Directed Nuclease1 (SDN1) and SDN2 developed products in many countries has made it more revolutionary technology for adoption in crop improvement. Designing the accurate guide RNA (gRNA) is the initial and most crucial step which decides the success of the gene editing. Though, the gene editing technique is widely used in crops, a detailed method on comprehensive designing of efficient gRNA in wheat is still lacking. By virtue of wheat being a hexaploid crop and having a large genome size with repetitive DNA, a tailor made strategy for designing the gRNA is very much crucial.The manuscript explains the comprehensive strategies and methods for efficient gRNA designing by considering physical, structural and expression of the target gene in genome and explains on-target and off-target effects of gRNA for its precise editing through CRISPR/Cas9 mediated SDN1 method of genome editing in wheat.The present manuscript is first of its kind to address the holistic approach starting from efficient gene selection, gRNA designing and post gRNA designing issues like gRNA stability, binding efficiency, and functionality for SDN1-CRISPR/Cas9 genome editing in wheat. This manuscript will acts as a ready reference for wheat researcher for effective gRNA designing for wheat improvement to meet the future food demand.