The CRISPR-based genome editing toolbox is packed with powerful refinements. The latest of these, prime editing (PE), was first reported in Nature in 2019 by David Liu’s group. Using a prime editing guide RNA that identifies the genomic target site and a catalytically inactive Cas9 fused to reverse transcriptase, prime editing can introduce small insertions and deletions in addition to single base changes.

Prime editors have been applied in animals and plants, but changes introduced at genomic sites other than the intended site (off-target effects) are a major hindrance in its application. Guide RNA (gRNA)-dependent off-target effects result from sequence similarities between the target site and other sites in the genome, whereas gRNA-independent off-target effects result from anomalous activity of CRISPR-based tools, such as deaminase, at non-target positions in the genome.

A new study from scientists at the Chinese Academy of Sciences (CAS) in Beijing demonstrates the specificity of PE in altering genomic DNA in plants establishing the potential of PE in agricultural biotechnology. The new study titled, “Genome-wide specificity of prime editors in plants,” and published in the journal Nature Biotechnology, reports a comprehensive and genome-wide analysis of the off-target effects of PE in rice plants.

As part of the study, the researchers first examined the mismatch tolerance of prime editors in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). The study reported that mismatches located in seed sequence regions of the spacer and near the nicking site of the Cas9 nickase reduced the frequency of PE implying high editing specificity.

The authors demonstrated designing pegRNAs with homology to fewer off-target sites is necessary for highly specific PE. They evaluate the frequencies of editing using 12 pegRNAs at 179 endogenous off-target sites containing mismatches and confirm that editing rates are extremely low (0.00–~0.23%).

The gRNA-independent effects induced by ectopic expression of functional elements in CRISPR-based tools detected previously with some base editors cannot be predicted in silico. Therefore, the research team led by Gao Caixia, PhD, professor at the Institute of Genetics and Developmental Biology (IGDB) of the CAS, used whole-genome sequencing to detect undesired genome-wide edits due to ectopic expression of the prime editors.

The authors confirm that prime editors do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions (indels) through whole-genome sequence analysis of 29 PE-treated rice plants.

The study also showed that ectopic expression of the Moloney murine leukemia virus reverse transcriptase (M-MLV RT) as part of the prime editor does not change the copy number of retrotransposons (a type of genetic component that copies and pastes itself into different genomic locations), the structure of telomeres or cause insertion of pegRNA or messenger RNA sequences into the genome. These results confirm that M-MLV RT, a core element of the prime editing machinery does not interfere with natural, endogenous reverse transcription mechanisms in the cell.