Ribozyme‐processed guide RNA enhances virus‐mediated plant genome editing

Abstract

In virus-induced gene-editing system, subgenomic promoters have been used to express guide RNAs (gRNAs). However, the transcription initiation site of the subgenomic promoters remains elusive. Here, we examined the sequence of gRNAs expressed by subgenomic promoters and found the variable length of overhangs at 5′-end of gRNAs. The overhangs at 5′-end of gRNA decrease the cleavage activity of SpCas9. To overcome this problem, we inserted hammerhead ribozyme between the subgenomic promoter and gRNA and confirmed that gRNAs with a precise 5′-end increase the editing efficacy in wild tobacco. This system will be widely used for editing target genes in plants with high efficiency.

RPS5A Promoter-Driven Cas9 Produces Heritable Virus-Induced Genome Editing in Nicotiana attenuata

“…S1 ). Although the mutation was observed in the infected leaves (19.7% ± 2.7%, Oh et al, 2021b ), we found no gene-edited seeds in the pooled M 1 seedlings of TRV2:gPDS-infected 35S:Cas9 ( Supplementary Fig. S1 , Oh et al, 2021b ).…”

“…To develop a VIGE system for N. attenuata , we first generated transgenic N. attenuata expressing SpCas9 under the control of the 35S promoter (35S:Cas9) ( Oh et al, 2021b ). A gRNA was designed to target the N. attenuata phytoene desaturase ( NaPDS ) gene and cloned into the TRV2 vector under the subgenomic promoter of the pea early-browning virus (TRV2:gPDS) ( Fig.…”

“…S2 ). Next, we inserted a hammerhead ribozyme sequence between the subgenomic promoter of TRV2 and NaPDS-targeted gRNA (TH:gPDS), because we found that TH:gPDS induced more indel mutations in the infected leaves than did T:gPDS ( Oh et al, 2021b ). A total of 123 M 1 progenies from TH:gPDS-infected 35S:Cas9 contained no mutations in the target site ( Supplementary Fig.…”

Microglial Replacement and COURIER or SPIT for Neuronal Gene Editing

“…Alternatively, the cargo could be a self-amplifying RNA (saRNA) vector [37]. An saRNA vector could enable the use of a CRISPR base editor, wherein a subgenomic promoter effectively replicates an sgRNA module but weakly replicates the proteinaceous component of the base editor [38,39]. As larger RNA molecules were packaged less efficiently in COURIER, but dual delivery was possible, a trans-amplifying RNA (taRNA) vector could be employed [40].…”

Editorial: CRISPR and alternative approaches

“…Ribozyme‐processed gRNAs are especially favorable to increase mutation frequency. [ 1 ] Trait‐improved cattle were generated through the genome‐editing technology CRISPR–Cas9, by targeting the Myostatin (MSTN) locus which led to offspring with a double‐muscling phenotype. [ 2 ] Chen and coworkers provided perspective on using CRISPR‐based platforms for rapid diagnostics, which could potentially revolutionize and expand our tool kits for tackling future pandemics.…”