Pre-validation strategies for CRISPR/Cas-based genome editing in plants: a critical analysis of in vitro RNP cleavage...

The advent of CRISPR/Cas-based genome editing has revolutionized crop improvement. However, the genome editing success rate remains a major challenge in many crops, especially those with challenging transformation protocols. We critically evaluate the integration of in vitro cleavage assays using naked target DNA and guide RNA-Cas9 nuclease (gRNA-Cas9) ribonucleoprotein (RNP) complexes as a pre-transformation validation step in genome editing workflows. We also compare other pre-validation methods with in vitro cleavage assays and present their advantages and limitations. In vitro assays can help directly confirm target cleavage and biochemically validate gRNA specificity. This strategy may facilitate the functional screening of gRNAs for plants with challenging and low transformation efficiency. In vitro assays can also reduce the unnecessary waste of resources and time associated with intensive transformation processes using non-specific gRNAs. Researchers can prioritize effective constructs based on the cleavage efficiency and specificity of the gRNAs. However, this assay may not guarantee simulation of the natural cellular environment for in vivo editing. We also present this pre-validation approach, which is particularly helpful for polyploid crops like wheat and cotton. In vitro cleavage assays offer a reliable pre-transformation screening step to identify highly active and specific gRNAs, thereby reducing resource-intensive transformation attempts. Future studies should integrate in vitro assays with advanced computational and in vivo validation tools to create a more predictive and efficient gRNA selection pipeline.