Abstract
Purpose :
The programmable double-strand break (DSB) inducing endonuclease CRISPR/Cas9 has emerged rapidly over the past years. Non-homologous end joining (NHEJ), microhomology mediated end joining (MMEJ) and homology directed repair (HDR) compete in the repair of the DSB, leading to unwanted insertions/deletions. Inactivation of one of the two catalytic active cleavage domains of the Cas9 molecule generates a nicking Cas9 (nCas9), which cleaves only one DNA strand, recruiting single strand repair molecules instead of DSB repair proteins. The Prime Edit (PE2) system uses a transcriptase fused to an nCas9 in combination with an extended prime edit guide RNA (pegRNA) containing a transcriptase template harboring the desired edit. The PE3 system uses an additional nicking guide RNA (ngRNA) targeting the opposite DNA strand. Employing the PE system, we aim to quantify the correction efficiency of the porcine Abca4 V1965X mutation (insertion of an adenine), using a cellular bioluminescence resonance energy transfer (BRET) based reporter system.
Methods :
Porcine Abca4 sequence containing the target mutation was cloned into the BRET reporter plasmid. Overall, 15 different pegRNAs were designed and cloned into a plasmid containing a U6 RNA promotor. HEK293-T cells were transfected with the Prime Edit Cas9, pegRNA (with/without additional nicking guide RNAs) and the BRET reporter. Correction of the target was analyzed by fluorescence microscopy, western blot and a BRET assay.
Results :
Prime editing using the PE2 system did not result in a significant correction as shown by western blot, fluorescence microscopy and the BRET assay. Additional nicking of the opposite DNA strand with the PE3 system generated a massive enhancement in correction activity of up to 91 ± 5 % measured with the BRET assay in dependence of pegRNA composition and ngRNA nicking position. Resulting correction measured with the BRET assay could be verified by fluorescence microscopy and western blot.
Conclusions :
We showed that the correction of an adenine insertion using the Prime Edit system can be reliably measured and quantified with the cellular BRET assay. Especially the PE3 can be a potential alternative to MMEJ- and HDR-based genome editing approaches, with comparably high correction rates, notably in post-mitotic cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.