Engineered pegRNAs improve prime editing efficiency.

Nat Biotechnol
Authors
Abstract

Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3' region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3' terminus of pegRNAs that enhance their stability and prevent degradation of the 3' extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3-4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3' structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3' motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.

Year of Publication
2021
Journal
Nat Biotechnol
Date Published
2021 Oct 04
ISSN
1546-1696
DOI
10.1038/s41587-021-01039-7
PubMed ID
34608327
Links
Grant list
U01Al142756 / Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)
RM1HG009490 / U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)
R01EB031172 / U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)
R35GM118062 / U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)