CRISPR-Cas13 mediated Knock Down inDrosophilacultured cells

Abstract

Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.

Precise genome engineering in Drosophila using prime editing

“…The advent of CRISPR has revolutionized genome editing in animal, plant, and microbe species (1). Researchers working with the model organism Drosophila melanogaster have utilized multiple CRISPR tools to study gene function, including Cas9, Cas12, and Cas13 (3)(4)(5)(6)(7)(8)(9). Drosophila is an important model because of its easy genetic manipulation, rich genomic resources, and usefulness to study human disease and disease transmission by insect vectors (10)(11)(12).…”

Efficient nanoparticle‐based CRISPR‐Cas13d induced mRNA disruption of an eye pigmentation gene in the white‐backed planthopper, Sogatella furcifera

“…Although a previous study developed a CRISPR‐Cas13d protocol for target gene disruption in insect cells (Viswanatha et al. , 2020), our approach incorporated a nanomaterial carrier (i.e. SPc) as a means of enhancing the in vivo efficiency of the CRISPR‐Cas13d system targeting living insects.…”

“…, 2020; Viswanatha et al. , 2020) and the high mortality of CRISPR‐Cas13d systems in mice that arises from collateral activity (Li et al. , 2023; Tong et al.…”

“…In this study, we developed a novel CRISPR-Cas13d RNA-degradation protocol to efficiently disrupt the functional role of the SfTO gene in WBPH eye pigmentation. Although a previous study developed a CRISPR-Cas13d protocol for target gene disruption in insect cells (Viswanatha et al, 2020), our approach incorporated a nanomaterial carrier (i.e. SPc) as a means of enhancing the in vivo efficiency of the CRISPR-Cas13d system targeting living insects.…”

“…The highest RNA-cleavage efficiencies were observed in the Cas13d group of enzymes, with the CasFX4 variant yielding a 90% knockdown rate. Similarly, heterologous expression of PspCas13b and RfxCas13d in cultured D. melanogaster S2 cells reduced the abundance of endogenous target transcripts (Viswanatha et al, 2020). The utility of the CRISPR-Cas13d system for functional genomics studies in nonmodel organisms, however, has yet to be as thoroughly explored.…”