Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA

• Published in: International journal of molecular sciences 2024-11, Vol.25 (23), p.12768
• Main Authors: Borovikova, Sofiia E, Shepelev, Mikhail V, Mazurov, Dmitriy V, Kruglova, Natalia A
• Format: Article
• Language: English
• Subjects: CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems; Cytoplasm; Efficiency; Enzymes; Gene Editing - methods; Gene therapy; Genome editing; HEK293 Cells; Humans; Jurkat Cells; Localization; Plasmids; Proteins; RNA polymerase; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; RNA, Guide, CRISPR-Cas Systems - genetics
• ISSN: 1661-6596; 1422-0067; 1422-0067
• DOI: 10.3390/ijms252312768

Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of 'NanoMEDIC' VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased and transgene knockout levels in 293 cells from 30% to 50-90% and raised the level of endogenous knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved knockout rates by up to 60-70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications.