Comprehensive benchmarking of genome editing quantification methods for plant applications.

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genomics

Background:

• Accurate quantification of CRISPR-Cas9 genome editing is vital for plant biotechnology and evaluating guide RNA (gRNA) performance.
• Current methods for measuring genome editing efficiency in plants lack standardization, hindering result comparability and reproducibility.
• Analyzing heterogeneous plant populations, especially from transient expression systems, requires sensitive and reliable editing detection techniques.

Purpose of the Study:

• To systematically evaluate and compare various experimental techniques for quantifying plant genome editing efficiency across a broad range of outcomes.
• To benchmark different quantification methods against targeted amplicon sequencing (AmpSeq) based on accuracy, sensitivity, and cost.
• To provide practical guidance for researchers on selecting appropriate genome editing quantification techniques for different plant applications.

Main Methods:

• Compared targeted amplicon sequencing (AmpSeq), PCR-restriction fragment length polymorphism (RFLP), T7 endonuclease 1 (T7E1) assays, Sanger sequencing with deconvolution algorithms, PCR-capillary electrophoresis/InDel detection by amplicon analysis (PCR-CE/IDAA), and droplet digital PCR (ddPCR).
• Quantified genome editing efficiency for 20 transiently expressed Cas9 targets in plants.
• Assessed methods based on accuracy, sensitivity, and cost, using AmpSeq as the reference standard.

Main Results:

• AmpSeq served as a reliable benchmark for assessing the accuracy and sensitivity of other genome editing quantification methods.
• Different techniques exhibited varying performance in detecting and quantifying CRISPR edits, highlighting the need for careful method selection.
• The study identified specific advantages and disadvantages for each tested method, including challenges encountered during optimization.

Conclusions:

• Standardization of genome editing quantification techniques is essential for improving the comparability and repeatability of plant research.
• The findings offer practical advice and guidelines for researchers to select the most suitable method for their specific plant genome editing applications.
• This comparative analysis aids both novice and experienced researchers in optimizing their experimental workflows for accurate CRISPR edit detection in plants.