Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events

Rapid and low-cost methods of detecting mutations and polymorphisms are crucial for genotyping applications including mutagenesis and gene editing. S1 family endonucleases such as T7E1, EndoV and CELI can potentially be used in enzymatic mismatch detection. Among them, CELI has been shown to be effective in detecting mutations in Targeting Induced Local Lesions IN Genomes (TILLING). However, current method of CELI purification from celery is laborious, and challenging for many non-biochemical laboratories, and the presence of post-translational modifications hinders efficient production of the enzyme in E. coli. Here, we report an efficient system for bulk production of enzymatically active CELI endonuclease through transient expression in a model plant Nicotiana benthamiana. We also optimized the reaction buffer, by additions of Mn²⁺ and DTT, with enhanced mismatch cleavage activity. Using the new CELI production and reaction system, we were able to routinely detect mismatches in 1/32 mixed mutant and wildtype DNA samples. We believe the newly established system has many applications in characterization of mutations occurred in natural variations, mutagenized populations and gene editing.

Keywords: Detections of mismatch and gene editing; Optimization of CELI digestion; Production of CELI endonuclease; Transient expression.