Abstract
CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.
Highlights
The development of CRISPR as a programmable genome engineering tool has revolutionized the life sciences by providing transformative applications for both medicine and biotechnology.[1]
Our results demonstrate that CasRx has some potential for programmable RNA targeting in flies, as we did observe some expected phenotypes for each target transcript, including green fluorescent protein (GFP), N, y and w
We did consistently observe both cellular toxicity from the ubiquitous expression of CasRx and dCasRx as we could not generate homozygous strains for either, and unexpected lethality and tissue necrosis, presumably due collateral off target effects which have been a feature previously observed for many CRISPR ribonucleases including CasRx.[2,4,7,8,9,51]
Summary
The development of CRISPR as a programmable genome engineering tool has revolutionized the life sciences by providing transformative applications for both medicine and biotechnology.[1] While much of the recent focus has been on exploiting CRISPR technologies to target DNA, recent findings that certain CRISPR systems can be programmed to target RNA have suggested new possibilities for CRISPR technologies in transcriptome engineering.[2,3,4] For example, one recent advancement was the engineering and biochemical characterization of Cas ribonuclease (CasRx) as a compact single-effector Cas enzyme that exclusively targets RNA with superior efficiency and specificity as compared to RNA interference (RNAi).[4] In human cells, CasRx demonstrated highly efficient on-target gene reduction with limited off-target activity, making it a potential tool for gene reduction This technology has yet to be comprehensively adapted for facile use in other systems ( see5), such as Drosophila melanogaster (flies), which are a common tool for exploring new biological questions and developing novel bioengineering tools in vivo. Non-RNAi-based techniques for reducing gene expression (without permanently altering the genome) in animals would provide for a more flexible technique to modulate gene expression in a biologically relevant way
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