Journal Article

Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage

Pääbo, Svante, Helmbrecht, Nelly, Kanis, Philipp, Riesenberg, Stephan, Maricic, Tomislav

2022

Overview

The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The ‘Genome-editing Optimized Locked Design’ (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit. Some DNA sequences are refractory to CRISPR-Cas9 cleavage, partially due to gRNA misfolding. Here the authors engineer gRNAs to prevent misfolding and further enhanced their stability by chemical modifications allowing robust genome editing regardless of target sequence.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 42/41 / 631/1647/1511 / 631/208/4041/3196 / 631/61/338 / Cell Line / Cleavage / CRISPR / CRISPR-Cas Systems - genetics / Deoxyribonucleic acid / Design modifications / Design optimization / DNA / Editing / Gene Editing / Gene sequencing / Genome / Genome editing / Genomes / gRNA / Humanities and Social Sciences / Humans / multidisciplinary / Nucleic Acid Conformation / Nucleotide sequence / Oligonucleotides - chemistry / RNA, Guide, CRISPR-Cas Systems - chemistry / RNA, Guide, CRISPR-Cas Systems - genetics / Science / Science (multidisciplinary) / Stability