Key result
AsCas12a-VLPs with CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50–90% and endogenous CXCR4 knockout in Jurkat T cells from 1% to up to 60-70%.
Why the study?
Nuclear localization of U6-driven sgRNA hinders efficient RNP formation and packaging into cytoplasmically assembling virus-like particles.
AsCas12a-VLPs with Pol II-transcribed crRNA efficiently mediate high levels of gene editing in mammalian cells, offering a promising tool for RNP delivery.
Does not support clinical use of AsCas12a-VLPs; leaves open further optimization and in vivo validation of crRNA delivery.
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 Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50–90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 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.
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Borovikova et al. (2024) studied this question. AsCas12a-VLPs with CMV-driven crRNA vs. SpCas9-VLPs was evaluated on Transgene knockout levels. AsCas12a-VLPs with CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50–90% and endogenous CXCR4 knockout in Jurkat T cells from 1% to up to 60-70%.
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