Fidelity and Delivery: Coupled Barriers in Plant CRISPR-Cas Genome Editing
TL;DR
This review proposes that these constraints are functionally coupled through the intracellular abundance, nuclear access, and chromatin residence time of active Cas-gRNA complexes, which could lead to convergent biochemical solutions spanning herbaceous and woody species.
Abstract
CRISPR-Cas platforms have transformed plant functional genomics, yet nucleases such as Cas9 and Cas12a remain constrained by two seemingly distinct limitations: imperfect target discrimination and inefficient intracellular delivery. Here, this review proposes that these constraints are functionally coupled through the intracellular abundance, nuclear access, and chromatin residence time of active Cas-gRNA complexes. Off-target activity arises from guide-target mismatch tolerance during R-loop formation, whereas rigid plant cell walls constrain the delivery of RNP and DNA cargo. These processes can become coupled when delivery limitations alter the concentration or duration of active Cas-gRNA exposure. When inefficient delivery is addressed through sustained or elevated nuclease expression, it can increase cumulative active Cas-gRNA exposure and may consequently increase off-target risk. Evidence from maize, rice, wheat, and carrot systems is synthesized and convergent biochemical solutions, including transgene-free RNP delivery, are outlined spanning herbaceous and woody species.