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

Frontiers in Plant Science
Satya Swathi NadakudutiDavid S Douches

Abstract

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 agaro...Continue Reading

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Citations

Dec 22, 2020·Synthetic Biology·Grace A MeakerThomas E Gorochowski

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Methods Mentioned

BETA
PCR
amplicon sequencing
electrophoresis
Assay
transfection
transfections
fluorescence activated cell-sorting
GUIDE-seq
amplicon-sequencing

Software Mentioned

TAL Effector Nucleotide Targeter
FLASH
FastQC
ImageJ
CRISPR RGEN
Nikon NIS Elements
GUIDE
Cutadapt
SAS

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