Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants

Abstract

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient non-homologous end-joining-mediated targeted insertion method was recently developed by combining CRISPR/SpCas9 gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5´overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5´ overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis), and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted two distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.

Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

“…Moreover, the multiplex PE system can serve as a valuable asset for functional genomics by enabling researchers to change the native gene sequence instead of relying on complementation assays with a transgenic approach. Multiple protein tagging with PE can help us understand complex gene-regulatory networks by tracking protein expression in native conditions ( Hua et al., 2022 ; Kumar et al., 2023 ).…”

Dual Activities of an X-family DNA Polymerase Regulate CRISPR-Induced Insertional Mutagenesis Across Species

“…Recent efforts to extend CRISPR-Cas9 genome editing into plants have confirmed the prevalence of 1-bp insertions in various plant species 9,[16][17][18] . While these findings suggested a potential extension of predictability observed in animals to plants, recent studies have also indicated that 1-bp insertion profiles may not be consistently predictable as proposed by the templated insertion model 9,16,17 . The existence of distinctive 1-bp insertion profiles and the underlying mechanism remains unclear.…”

Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress‐related proteins

“…Heritable GT can be enhanced by specifically expressing CAS9 in egg cells or embryos (Wang et al, 2015; Wolter et al, 2018). Note that while we focus here on procedures involving double strand breaks and homology‐directed repair, recent work has also reported GT methods that utilise NHEJ to insert new sequence (e.g., Cermak, 2021; Kumar et al, 2023).…”