CRISPR/Cas-mediated in planta gene targeting: current advances and challenges

Abstract

We can use gene targeting (GT) to make modifications at a specific region in a plant's genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant GT. Several studies have recently demonstrated improvements in GT efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified GT-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated GT in plants and discuss potential efficiency improvements. Increasing the efficiency of GT technology will help us pave the way for increased crop yields and food safety in environmentally friendly agriculture.

Prime editing: Mechanism insight and recent applications in plants

“…Even today, not all types of base transversion are readily accessible with BEs (Chen and Liu, 2023 ). Moreover, the efficiency of GT was not sufficiently high at many loci (Guzmán‐Benito et al ., 2023 ; Vu et al ., 2019 ), and the introduced double‐stranded breaks (DSBs) may lead to unwanted impacts on the genome and the viability of targeted cells. Anzalone and coworkers thought that the 3′‐hydroxyl generated by nCas9 (H840A) could prime the RT reaction with a desired edit‐carrying extension of the single‐guide RNA (sgRNA) as the RNA template (Anzalone et al ., 2019 ).…”