Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

• Main Authors: Satya Swathi Nadakuduti, Colby G. Starker, Dae Kwan Ko, Thilani B. Jayakody, C. Robin Buell, Daniel F. Voytas, David S. Douches
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2019-02-01
• Series: Frontiers in Plant Science
• Subjects: genome-editing; CRISPR/Cas9; TALENs; protoplasts; double-stranded oligodeoxynucleotides; NHEJ
• Online Access: https://www.frontiersin.org/article/10.3389/fpls.2019.00110/full

Genome-editing is being implemented in increasing number of plant species using engineered sequence specific nucleases (SSNs) such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9), Transcription activator like effector nucleases (TALENs), and more recently CRISPR/Cas12a. As the tissue culture and regeneration procedures to generate gene-edited events are time consuming, large-scale screening methodologies that rapidly facilitate validation of genome-editing reagents are critical. Plant protoplast cells provide a rapid platform to validate genome-editing reagents. Protoplast transfection with plasmids expressing genome-editing reagents represents an efficient and cost-effective method to screen for in vivo activity of genome-editing constructs and resulting targeted mutagenesis. In this study, we compared three existing methods for detection of editing activity, the T7 endonuclease I assay (T7EI), PCR/restriction enzyme (PCR/RE) digestion, and amplicon-sequencing, with an alternative method which involves tagging a double-stranded oligodeoxynucleotide (dsODN) into the SSN-induced double stranded break and detection of on-target activity of gene-editing reagents by PCR and agarose gel electrophoresis. To validate these methods, multiple reagents including TALENs, CRISPR/Cas9 and Cas9 variants, eCas9(1.1) (enhanced specificity) and Cas9-HF1 (high-fidelity1) were engineered for targeted mutagenesis of Acetolactate synthase1 (ALS1), 5-Enolpyruvylshikimate- 3-phosphate synthase1 (EPSPS1) and their paralogs in potato. While all methods detected editing activity, the PCR detection of dsODN integration provided the most straightforward and easiest method to assess on-target activity of the SSN as well as a method for initial qualitative evaluation of the functionality of genome-editing constructs. Quantitative data on mutagenesis frequencies obtained by amplicon-sequencing of ALS1 revealed that the mutagenesis frequency of CRISPR/Cas9 reagents is better than TALENs. Context-based choice of method for evaluation of gene-editing reagents in protoplast systems, along with advantages and limitations associated with each method, are discussed.