Chimeric oligonucleotides combining guide RNA and single-stranded DNA repair template effectively induce precision gene editing.

The ability to precisely alter the genome holds immense potential for molecular biology, medicine and biotechnology. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) into a genomic editing tool has vastly simplified genome engineering. Here, we explored the use of chemically synthesized chimeric oligonucleotides encoding a target-specific crRNA (CRISPR RNA) fused to a single-stranded DNA repair template for RNP-mediated precision genome editing. By generating three clinically relevant oncogenic driver mutations, two non-stop extension mutations, an FGFRi resistance mutation and a single nucleotide change, we demonstrate the ability of chimeric oligos to form RNPs and direct Cas9 to effectively induce genome editing. Further, we demonstrate that the polarity of the chimeric oligos is crucial: only chimeric oligos with the single-stranded DNA repair template fused to the 3'-end of the crRNA are functional for accurate editing, while templates fused to the 5'-end are ineffective. We also find that chimeras can perform editing with both symmetric and asymmetric single-stranded DNA repair templates. Depending on the target locus, the editing efficiency using chimeric RNPs is similar to or less than the efficiency of editing using the bipartite standard RNPs. Our results indicate that chimeric RNPs comprising RNA-DNA oligos formed from fusing the crRNA and DNA repair templates can successfully induce precise edits. While chimeric RNPs do not display an advantage over standard RNPs, they nonetheless represent a viable approach for one-molecule precision genome editing.

Universal Template-Assisted, Cloning-free Method for the Generation of Small RNA-Expressing Dumbbell-Shaped DNA Vectors.

Dumbbell-shaped DNA minimal vectors represent genetic vectors solely composed of the gene expression cassette of interest and terminal closing loop structures. Dumbbell vectors for small hairpin RNA or microRNA expression are extremely small-sized, which is advantageous with regard to cellular delivery and nuclear diffusion. Conventional strategies for the generation of small RNA-expressing dumbbell vectors require cloning of a respective plasmid vector, which is subsequently used for dumbbell production. Here, we present a novel cloning-free method for the generation of small RNA-expressing dumbbell vectors that also does not require any restriction endonucleases. This new PCR-based method uses a universal DNA template comprising an inverted repeat of the minimal H1 promoter and the miR-30 stem. The sequences coding for small RNA expression are introduced by the PCR primers. Dumbbells are formed by denaturing and reannealing of the PCR product and are covalently closed using ssDNA ligase. The new protocol generates plus- and/or minus-strand dumbbells, both of which were shown to trigger efficient target gene knockdown. This method enables fast, cheap production of small RNA-expressing dumbbell vectors in a high throughput-compatible manner for functional genomics screens or, as dumbbells are not prone to transgene silencing, for knockdown studies in primary cells.