Name: ubiquitous and tissue-specific rna targeting in drosophila melanogaster using crispr/casrx
Uploaded: 2021-02-05T15:00:00
Duration: 6 min 37 s
Description: Watch this Scientific Journal Video about Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx at JoVE.com

This work was supported in part by funding from a DARPA Safe Genes Program Grant (HR0011-17-2-0047), and NIH awards (R21RAI149161A, DP2AI152071) awarded to O.S.A.

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O.S.A is a founder of Agragene, Inc., has an equity interest, and serves on the company's Scientific Advisory Board. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. All other authors declare no competing interests.

With three different application designs of the CasRx system, this work demonstrated&#160;in vivoprogrammable RNA targeting in flies. The different strategies cater to different project needs, such as endogenous versus exogenous gene targeting and ubiquitous versus tissue-specific RNA targeting. The effects of RNA targeting included target gene specific phenotypic changes, target RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner.
One of the key factors in successful customizing of the CasRx system is the design of gRNAs. Specifically, the following advice shall be heeded: the target sequence is around 30 nucleotides in length, the length of poly-U stretches in the target sequence is 4 base pairs or less, the target sequence GC content is in the range of 30% - 70%, the target sequence is not predicted to form strong RNA hairpin structures, and the target sequence contains minimal predicted RNA secondary or tertiary structure5.
In addition to the gRNA designs, the fly genetics step in each protocol is also critical in a successful implementation. The presence or lack of the defined phenotypes passed down from the parents in the progenies are important for identifying and quantifying phenotypes induced by the CasRx system in the transheterozygous progenies. Also, setting up control crosses using the dCasRx flies in parallel are also helpful in the ruling out non-specific phenotypes in the transheterozygous progenies.
It is worth noting that these results revealed toxicity issue introduced by ubiquitously expressing CasRx and dCasRx protein in the fly, a limitation of CasRx system. Ubiquitous expression of CasRx or dCasRx under the Ubiq promotor alone, without gRNAs, came with nontrivial fitness costs, as neither Ubiq-CasRx nor Ubiq-dCasRx flies could be established as homozygous lines. On the contrary, UASt-CasRx and UASt-dCasRx flies can be established as healthy homozygous stocks, though due to the design of the cross scheme they were kept as double-balanced stocks, a fact that supports the existence of toxicity induced by ubiquitous CasRx protein expression. Another piece of supporting evidence is that in control experiments involving dCasRx, which is catalytically inactive, the percentages of flies carrying both dCasRx and gRNA constructs out of the total number of flies in the F1 generation were consistently lower than 50%, the ratio expected based on Mendelian genetics if no dCasRx-associated toxicity was present. This indicated that ubiquitously expressing dCasRx, along with gRNAs, induces toxicity in the fly, resulting less than expected inheritance ratio. The inheritance ratios of transheterozygous UASt-dCasRx, gRNA, GAL4 flies followed Mendelian genetics, which again suggests the toxicity induced specifically by ubiquitous expression of CasRx and dCasRx proteins. Toxicity in CRISPR/Cas system is not new. High amounts of Cas9 protein has been shown to be toxic in several organisms, including flies29,30,31,32. A recent study has developed a customized GAL4/UAS system that can tune the amount of Cas9 protein expressed in flies by adding an open-reading frame of varying length between the UAS sequence and the Cas9 sequence in the UAS-Cas9 construct33. Therefore, it is worth exploring ways to reduce CasRx-induced toxicity by tuning the CasRx protein expression level.
Other than the toxicity induced by ubiquitous expression of CasRx and dCasRx proteins, the results also showed lethality linked to CasRx system&#39;s non-specific collateral off-target effects, a feature of many CRISPR systems1, 2, 7, 34. In some of the CasRx and non-essential gene gRNA-expressing double or triple transheterozygous flies, for example when targeting Notch, the transheterozygous CasRx flies have significantly lower levels of survival rate compared to the transheterozgyous dCasRx flies. In the RNA-seq analysis of these CasRx and gRNA-expressing transheterozygous flies, both the reduction of target gene transcript levels and the reduction of non-target gene transcripts were observed. These collateral effects were CasRx-dependent and target-dependent, as they were only observed in transheterozygous flies expressing both the CasRx protein and the gRNA. It is worth pointing out that one of the target genes, white, showed only a limited, non-statistically significant reduction in transcripts when the white gene was targeted by CasRx, which was in contrast to the clear pigment reduction phenotype. It is hypothesized that this may be due to the fact that 1) the timing of RNA-seq sample collection was not well aligned with the timing when the white gene reach its peak expression during early development, and 2) the localized expression of the white gene in the eyes makes it challenging to collect the relevant tissues during early development phase when only whole-body sample collection is feasible. To reduce collateral activity in the CasRx system, future studies are called for to fully understand the mechanisms underlying the off-target phenomenon system at the organismal level.
Interestingly, a recent study35 describing RNA-targeting Cas13 tools in flies appeared to ameliorate the general toxicity associated with CasRx expression, for several possible reasons. Firstly, the authors recoded the Cas13 transgenes to optimize expression in Drosophila and utilized a more weakly-expressing promoter (actin 5C) as compared to the ubiquitin promoter used in the present study, likely leading to lower levels of Cas13 expression and thus less toxicity. Indeed, this is supported by the observations that UASt-driven CasRx and dCasRx expression was not, by itself, toxic, as this study (and the authors in 35) did not observe any obvious lethality in UASt-CasRx flies. Furthermore, these authors encoded their gRNAs differently compared to this study, which may have affected their expression and reduced the toxicity of the system in transheterozygous Cas13/gRNA flies. For example, in their study two gRNAs were expressed using the U6:3 promoter and flanked by tRNAs to enable gRNA processing upon tRNA maturation without requiring CasRx35. Conversely, in this study, the gRNAs were encoded as arrays targeting up to 4 locations per gene and mimicking the endogenous Cas13 array structure found in bacteria, which requires the Cas13 enzyme to process each gRNA. These different approaches may have led to differences in gRNA expression levels and other factors that may have inherent effects on the toxicity of the whole system. Finally, Huynh et al. targeted different genes than those targeted in the present study, which result in differences in target-Cas/gRNA interaction and collateral activity and may have effects on the observed levels of lethality. These differences in observed toxicity warrant further investigation to identify ways that the overall systems can be improved.
Overall, this study is the first demonstration of a functional genetically encoded programmable RNA-targeting Cas system in D. melanogaster, albeit further optimization of the CasRx system (in line with what is reported35) will be required to further reduce the off-target-associated lethality and increase the efficacy of CasRx on-target cleavage. RNA-targeting with Cas enzymes is a rapidly evolving field with many potential applications ranging from insect vector control to therapeutic usages1,2,3,4,5,6,7, and this protocol offer a starter package for anyone interested in designing their first CasRx system in flies, while being compatible with customization and further optimization of the system. The examples presented here demonstrate a range of results one may encounter during implementation of this system in vivo and may serve as benchmarks for other users in evaluating the performance of CasRx system in their applications.

Since the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies, much of the focus in this field has been on DNA editing, which offers transformative applications in medicine and biotechnology1. Permanent alteration of DNA sequences, however, is not always desired due to ethical considerations. In light of this, recent studies began developing CRISPR-based tools for targeting RNA and demonstrated that CRISPR technologies can indeed be used for RNA-targeting in a variety of biological systems2,3,4,5,6,7. In many of these systems tested, the current widely used approach for targeting RNA and transcript reduction is RNA interference (RNAi), which is far from perfect, often exhibiting varied efficacy and high off-target activity when used in vivo8,9,10,11,12,13,14,15,16,17. Therefore, given the status of these technologies, it is worth further exploring the potentials of CRISPR-based tools for RNA targeting.
One notable recent study reported that ribonuclease CasRx, a member of the Cas13d class, can efficiently reduce gene transcript levels in human cell culture and possesses an attractive off-target profile4. This finding led to the question of whether this new ribonuclease can maintain its efficacy and low off-target rate for RNA targeting at the organismal level. A recent study addressed this question by showing that the CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster5.
To streamline usability of this recently published approach, this protocol details the methods from this recent work, which consists of three main parts: 1) ubiquitous in vivo&#160;RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specificin vivo RNA targeting using a three-component CasRx system.
Guide RNAs (gRNA) targeting different target genes under the control of a ubiquitous promoter were designed and fly lines expressing these gRNA-containing constructs were generated. CasRx constructs under the control of either a ubiquitous promoter, or a conditional upstream activation sequence (UASt) promoter activatable by the GAL4 transcription factor, were also designed and fly lines harboring these CasRx-containing constructs generated. Catalytically inactive CasRx constructs, dCasRx, were designed and used as negative controls. Ubiquitous RNA targeting in flies is achieved by crossing gRNA-expressing fly lines with ubiquitously CasRx-expressing fly lines. The progeny expressing both the gRNA construct targeting a specific gene transcript and the CasRx protein has a ubiquitous reduction of targeted gene transcripts. Tissue-specific RNA targeting in flies is achieved by first crossing gRNA-expressing flies with UASt-CasRx expressing flies, obtaining transheterozygous flies carrying both gRNA and UASt-CasRx constructs. Such flies in turn are crossed with tissue-specific GAL4-expressing flies, resulting in the generation of tissue-specific CasRx expression and RNA-targeting in flies.
The programmable nature of the CasRx system offers the possibility of customization and optimization to help achieve high efficacy and low off-target activity for in vivo RNA targeting. Potential applications of CRISPR-based RNA-targeting are numerous, including replacing RNAi in the laboratory and contributing to insect vector control in the wild. Of the latter, one of the global unmet needs is the development of efficient tools to combat infections of RNA viruses transmitted via mosquitos. Many RNA viruses, such as dengue, Zika, and chikungunya virus, are transmitted via mosquitoes, affecting human health, and contributing mortality. Many proposals for engineering mosquito populations with virus resistance for disease prevention have been made; however, no current technology is able to make mosquitoes simultaneously resistant to all significant RNA viruses18,19,20,21,22,23. RNA-targeting Cas systems may provide a starting point for such a technology by enabling a programmable platform for targeting all mosquito-borne RNA viruses.

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	<tbody>
		<tr>
			<td>100% Grape Juice</td>
			<td>Welch Foods Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Active Dry Yeast (908g)</td>
			<td>Red Star Yeast Company, LLC</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMC4500 color camera</td>
			<td>Leica Microsystems</td>
			<td>DMC4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>GAL4-GMR flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>29967</td>
			<td></td>
		</tr>
		<tr>
			<td>GAL4-y flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>44373</td>
			<td></td>
		</tr>
		<tr>
			<td>Glomax 20/20 Luminometer</td>
			<td>Promega</td>
			<td>E5331</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina HiSeq2500 Sequencer</td>
			<td>Illumina, Inc.</td>
			<td>HiSeq2500</td>
			<td></td>
		</tr>
		<tr>
			<td>M165FC fluorescent stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>M165FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop OneC UV-vis spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>NDONEC-W</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra II RNA Library Prep Kit</td>
			<td>New England Biolabs, Inc.</td>
			<td>E7770</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 112686</td>
			<td>Addgene</td>
			<td>112686</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 112688</td>
			<td>Addgene</td>
			<td>112688</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132416</td>
			<td>Addgene</td>
			<td>132416</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132417</td>
			<td>Addgene</td>
			<td>132417</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132419</td>
			<td>Addgene</td>
			<td>132419</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132420</td>
			<td>Addgene</td>
			<td>132420</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132421</td>
			<td>Addgene</td>
			<td>132421</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132422</td>
			<td>Addgene</td>
			<td>132422</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132425</td>
			<td>Addgene</td>
			<td>132425</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 132426</td>
			<td>Addgene</td>
			<td>132426</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 133304</td>
			<td>Addgene</td>
			<td>133304</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid # 164586</td>
			<td>Addgene</td>
			<td>164586</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid #132418</td>
			<td>Addgene</td>
			<td>132418</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pJFRC81</td>
			<td>Addgene</td>
			<td>36432</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction endonucleases AscI</td>
			<td>New England Biolabs Inc.</td>
			<td>R0558L</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction endonucleases NotI</td>
			<td>New England Biolabs Inc.</td>
			<td>R0189L</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction endonucleases PacI</td>
			<td>New England Biolabs Inc.</td>
			<td>R0547L</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction endonucleases PstI</td>
			<td>New England Biolabs Inc.</td>
			<td>R0140L</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction endonucleases SwaI</td>
			<td>New England Biolabs Inc.</td>
			<td>R0604L</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction endonucleases XbaI</td>
			<td>New England Biolabs Inc.</td>
			<td>R0145L</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico Kit for Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbo DNase</td>
			<td>Invitrogen</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>U6-3:4-gRNA-Fluc flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84125</td>
			<td></td>
		</tr>
		<tr>
			<td>U6-3:4-gRNA-GFP; OpIE2-GFP flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84986</td>
			<td></td>
		</tr>
		<tr>
			<td>U6-3:4-gRNA-N flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84122</td>
			<td></td>
		</tr>
		<tr>
			<td>U6-3:4-gRNA-w flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84124</td>
			<td></td>
		</tr>
		<tr>
			<td>U6-3:4-gRNA-y flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84123</td>
			<td></td>
		</tr>
		<tr>
			<td>UASt-CasRx flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84121</td>
			<td></td>
		</tr>
		<tr>
			<td>UASt-dCasRx flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiq-CasRx flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84118</td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiq-dCasRx flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84119</td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiq-Firefly-T2A-eGFP-Ubiq-Renilla flies</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>84127</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean Gel DNA Recovery Kits</td>
			<td>Zymo Research Corporation</td>
			<td>D4007</td>
			<td></td>
		</tr>
	</tbody>
</table>

ubiquitous and tissue-specific rna targeting in drosophila melanogaster using crispr/casrx

CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It is recently reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous&#160;in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.

Ubiquitous in vivo RNA Targeting Using a Two-Component CasRx System 
The F1 transheterozygous flies expressing both the Ubiq-CasRx and the gRNA (targeting both endogenous and exogenous genes) constructs showed marked phenotypes compared to the control flies expressing the Ubiq-dCasRx and gRNA constructs (Figure 2 and Figure 4). Specifically, the transheterozygous CasRx flies have significantly lower levels of survival rate compared to the transheterozgyous dCasRx flies, indicating toxicity of the Ubiq-CasRx system (Figure 2A and Figure 4A). It is worth noting that both transheterozygous CasRx and dCasRx flies have less than 50% inheritance rate, which is the expected ratio based on Mendelian genetics. Of the three target genes, the Ubiq-CasRx/+; U6-gRNAN/+ flies and Ubiq-CasRx/+; U6-gRNAy/+ flies are non-viable (0% inheritance) and did not grow beyond the second instar larvae stage (Figure 2A-2B). The surviving Ubiq-CasRx/+; U6-gRNAw/+ flies, the inheritance of which was 12.9%, showed a distinct fully-penetrant white-eyed phenotype (Figure 2B). In addition to observable traits associated with CasRx, we were able to confirm significant reduction of target gene transcripts for 3 target genes: Notch, yellow, and GFP (Figure 2E-2G). Reduction of white gene transcripts was observed in Ubiq-CasRx/+, U6-gRNAw/+ flies, compared to the control Ubiq-dCasRx/+, U6-gRNAw/+ flies, though the reduction was not statistically significant (Figure 2E - 2F). Evidence of off-target activity induced by CasRx was found when comparing the differentially expressed transcripts between samples from CasRx-expressing flies and samples from dCasRx-expressing flies (Figure 2E, 2G). The number of non-target transcripts significantly differentially expressed are as follows: white, 253 (1.4% of total transcripts); Notch, 300 (1.7%); yellow, 41 (0.23%); GFP, 5,880 (33%) (Figure2G). Out of the total 17,779 different transcripts, 6 non-target transcripts were significantly differentially expressed in all 4 groups of samples. One of the 6 transcripts identified was Gadd45, a gene involved in apoptosis and cellular arrest in flies, raising the possibility that the enzymatic action of CasRx may either directly trigger cellular apoptosis or indirectly trigger misexpression of other genes, which in turn leads to apoptosis. Finally, it is worth noting that the Ubiq-CasRx and Ubiq-dCasRx flies were not established as homozygous stocks, presumably due to toxicity conferred by high ubiquitous expression. As a result, heterozygous Ubiq-CasRx/CyO and Ubiq-dCasRx/CyO flies were used for crossing with homozygous gRNA fly lines. In sum, the two-component Ubiq-CasRx system is able to achieve ubiquitous RNA targeting for both endogenous and exogenous targets resulting in observable phenotypes and transcript reduction. These results also showed that CasRx-mediated RNA targeting may introduce toxicity in vivo.
Ubiquitous in vivo exogenous RNA Targeting Using a Three-Component CasRx System 
The results from the two-step cross showed that despite the exogenous nature of the target gene (i.e., Fluc), expressing all three transgenes in F2 triple transheterozygotes (Ubiq-CasRx/+; gRNAFluc/Ubiq-Fluc-Ubiq-Rluc) resulted in 100% lethality compared to control crosses involving Ubiq-dCasRx, where no lethality was observed in the F2 triple transheterozygotes (Ubiq-dCasRx/+; gRNAFluc/Ubiq-Fluc-Ubiq-Rluc) (Figure 3B-C). More specifically, only the combination of all three transgenes (Ubiq-CasRx/+; gRNAFluc/Ubiq-Fluc-Ubiq-Rluc) resulted in 100% lethality (Figure 3B and D), while (Ubiq-CasRx/+; gRNAFluc/TM6) and (Ubiq-CasRx/+; Ubiq-Fluc-Ubiq-Rluc/TM6) genotypes were viable and lacked phenotypes with their inheritance rates matching the expected Mendelian transmission rates, suggesting that the availability of the target sequence (i.e., firefly luciferase) in combination with Ubiq-CasRx/+ and the gRNAFluc&#160;is what resulted in the observed lethality phenotypes, presumably stemming from the collateral activity of Cas13 enzymes2,8. In addition, no distinguishable phenotypes or dramatic influence on inheritance in F1 transheterozygotes (Ubiq-CasRx/+; gRNAFluc/+ or Ubiq-CasRx/+; Ubiq-Fluc-Ubiq-Rluc/+) were observed compared to Ubiq-dCasRx controls (Ubiq-dCasRx/+; gRNAFluc/+ or Ubiq-dCasRx/+; Ubiq-Fluc-Ubiq-Rluc/+) (Figure 3B), indicating that a catalytically active enzyme is essential to obtain the lethality phenotypes observed. Furthermore, Fluc and Rluc expression levels in flies of all viable genotypes did not show significant reduction in Fluc expression in the Ubiq-dCasRx triple transheterozygotes (Ubiq-dCasRx/+; gRNAFluc/Ubiq-Fluc-Ubiq-Rluc) compared to dual luciferase reporter controls. This suggests that Fluc protein expression levels were not reduced by dCasRx targeting (Figure 3D). Taken together, the common lethality phenotype in the two different CasRx-mediated ubiquitous RNA targeting experiments indicate that when used on tissues ubiquitously, CasRx-mediated RNA targeting can be toxic to the organism.
Tissue-specific in vivo RNA Targeting Using a Three-Component CasRx System 
The high level of toxicity observed in ubiquitous RNA targeting experiments prompted us to explore the tissue-specific RNA targeting using a three-component CasRx system design detailed in the methods section. Indeed, the level of toxicity observed was reduced when the overall CasRx expression was lowered using the UASt promoter compared to that of the Ubiq promoter, this is exemplified in three aspects: 1) the UASt-CasRx and UASt-dCasRx lines were maintained as homozygous lines, though based on the two-step cross scheme double balanced UASt-CasRx and UASt-dCasRx lines were used to perform the crosses, 2) all F2 generation dCasRx triple transheterozygous inheritance rates matched the expected 25% Mendelian inheritance rate, and 3) the F2 generation CasRx triple transheterozygous lethality phenotype was moderately reduced. In the white targeting experiment, of the 25% Mendelian inheritance rates expected in the F2 triple transheterozygotes, only 0.57% viable adult flies (UASt-CasRx/+; gRNAw/GMR-Gal4) were observed, all of which displayed severe eye specific pigmentation and morphology phenotypes (Figure 4A and 4B). For the white-targeting cross, the CasRx-expressing triple transheterozygous F2 inheritance rate was significantly lower than that of the dCasRx-expressing triple transheterozygous control group (27.6%) (Figure 4A). In the Notch targeting experiment, CasRx-expressing triple tranheterozygous carrying all three transgenes were 100% lethal, while the dCasRx control inheritance rate was 29.3% (Figure 4A). In the yellow targeting experiment, F2 triple transheterozygous CasRx-expressing, gRNAy, and y-GAL4 showed marginal chitin pigment reduction as small patches of yellow cuticle on the thorax and abdomen with an inheritance rate of 2.67%, much lower than that of the dCasRx control group (25.2%) (Figure4A). All dCasRx control triple transheterozygous flies did not present obvious phenotypes as the CasRx-expressing flies, indicating that catalytic activity of CasRx contributed to the phenotypes observed. The low inheritance rate in the CasRx triple transheterozygous group suggested that two sources of toxicity exist in CasRx RNA targeting: one is associated with high expression of CasRx, the toxicity of which was reduced by restrictive CasRx expression, the other is associated with the collateral activity. Taken together, these results showed that the CasRx system can achieve tissue-specific in vivo RNA targeting by leveraging the classical Gal4/UASt system and in the meantime reduce the toxicity. However, toxicity and occasional lethality phenotypes were still observed at a lower level of severity compared to that of the ubiquitous approaches, indicating that collateral cleavage activity is associated with toxicity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62154/62154fig01.jpg" /> 
Figure 1: General overview of RNA targeting using a Cas13D system. (A) Schematics of the one-step genetic cross in the ubiquitous in vivo RNA targeting using a two-component CasRx system. (B) Schematics of a two-step genetic cross in the ubiquitous&#160;in vivo exogenous RNA targeting using the three-component CasRx system. (C) Schematics of a two-step genetic cross in the tissue-specific in vivo RNA targeting using a three-component CasRx system. <a href="https://www.jove.com/files/ftp_upload/62154/62154fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62154/62154fig02.jpg" /> 
Figure 2: Ubiquitous in vivo RNA targeting using a two-component CasRx system (reprinted5). (A) Total inheritance percentages of transheterozygous flies inheriting Ubiq-CasRx (or Ubiq-dCasRx) and gRNAs. Blue shading in the box plot indicates phenotype penetrance. (B) Phenotypes of transheterozygous flies. Arrows indicate tissue necrosis in the eye. Black and white fly marked with &#39;&#39;X&#39;&#39; represents lethality. (C) Total inheritance percentages of transheterozygous flies of bidirectional crosses between Ubiq-CasRx (or Ubiq-dCasRx) and gRNAGFP-OpIE2-GFP flies. M, maternal inheritance of CasRx; P, paternal inheritance of CasRx. (D) F1 larvae progenies in the paternal cross. (E) Transcripts&#39; maximum a posteriori estimates for the logarithmic fold change. DESeq2 pipeline was used. (F) Transcripts per million (TPM) targeted with CasRx or dCasRx. (G) CasRx-depentent differentially expressed transcript percentage of transcripts. <a href="https://www.jove.com/files/ftp_upload/62154/62154fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62154/62154fig03.jpg" /> 
Figure 3: Ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system. (A) Schematics of the two-step genetic cross. (B) Total inheritance percentages for all genotypes emerging in the F2 generation. Inheriting all three transgenes (Ubiq-CasRx, Ubiq-Fluc-Ubiq-Rluc, and gRNAFLuc) in F2 progeny resulted in 100% lethality and was significantly lower compared to the Ubiq-dCasRx triple transheterozygotes control group (p = 0.001, t-test). (C) Carrying Ubiq-CasRx/gRNAFluc alone or Ubiq-CasRx and Ubiq-Fluc-Ubiq-Rluc alone did not lead to severe lethality, and inheritance ratios between Ubiq-CasRx and Ubiq-dCasRx transheterozygotes were not significantly different (p = 0.41 and p = 0.51, respectively, t-test). (D) Luciferase ratios normalizing Fluc readings to Rluc readings. Triple transheterozygous flies expressing Ubiq-CasRx, Ubiq-Fluc-Ubiq-Rluc, gRNAFLuc were embryonic lethal, which was represented by a fly with an &#34;X&#34;, and as a result luciferase expression was not measured. Fluc/Rluc ratio of Ubiq-CasRx/+, Ubiq-Fluc-Ubiq-Rluc/TM6, Stb transheterozygotes was significantly lower than that of the other Ubiq-Fluc-Ubiq-Rluc-expressing groups (p = 1.2e-06 or lower, t-test). The results from the gRNAFLuc-only group were significantly lower than that of all other groups (p = 1.2e-06 or lower, t-test). <a href="https://www.jove.com/files/ftp_upload/62154/62154fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62154/62154fig04.jpg" /> 
Figure 4: Tissue-specific in vivo RNA targeting using a three-component CasRx system (reprinted5). (A) Total inheritance percentage of triple transheterozygous flies carrying three transgenes (UASt-CasRx or UASt-dCasRx, gRNAs, and Gal4-driver. (B) Phenotypes of the triple transheterozygous flies. The white arrow indicates chitin pigment reduction in the thorax. Black and white fly marked with &#39;&#39;X&#39;&#39; represents lethality. <a href="https://www.jove.com/files/ftp_upload/62154/62154fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Construct</td>
			<td>Description</td>
			<td>Primer</td>
			<td>Primer Sequence (5&#8217; to 3&#8217;)</td>
			<td>PCR Template</td>
		</tr>
		<tr>
			<td rowspan="2">OA-1050E</td>
			<td rowspan="2">CasRx</td>
			<td>1050E.C3</td>
			<td>TACTAATTTTCCAC 
			ATCTCTATTTTGAC 
			CCGCAGATTAATTA 
			ATGAGCCCCAAGA 
			AGAA</td>
			<td rowspan="2">pNLS-RfxCas13d-NLS-HA (pCasRx)</td>
		</tr>
		<tr>
			<td>1050E.C4</td>
			<td>CAATTGATTTGTTA 
			TTTTAAAAACGATT 
			CATTCTAGCTAGCT 
			TAAGCGTAATCTGG 
			AACA</td>
		</tr>
		<tr>
			<td rowspan="2">OA-1050R</td>
			<td rowspan="2">dCasRx</td>
			<td>1050E.C3</td>
			<td>TACTAATTTTCCAC 
			ATCTCTATTTTGAC 
			CCGCAGATTAATTA 
			ATGAGCCCCAAGA 
			AGAA</td>
			<td rowspan="2">pNLS-dRfxCas13d-NLS-HA (pdCasRx)</td>
		</tr>
		<tr>
			<td>1050E.C4</td>
			<td>CAATTGATTTGTTA 
			TTTTAAAAACGATT 
			CATTCTAGCTAGCT 
			TAAGCGTAATCTG 
			GAACA</td>
		</tr>
		<tr>
			<td rowspan="4">OA-1050L</td>
			<td rowspan="2">UASt promoter</td>
			<td>1041.C9</td>
			<td>GCGGGTTCTCGA 
			CGGTCACGGCGG 
			GCATGTCGACGC 
			GGCCGCAACCAA 
			CAACACTAGTAG</td>
			<td rowspan="2">pJFRC81</td>
		</tr>
		<tr>
			<td>1041.C11</td>
			<td>CTGGCCTCCACC 
			TTTCTCTTCTTCT 
			TGGGGCTCATGT 
			TTAAACCCAATT 
			CCCTATTCAGA</td>
		</tr>
		<tr>
			<td rowspan="2">CasRx</td>
			<td>1050L.C1</td>
			<td>AATACAAGAAGA 
			GAACTCTGAATA 
			GGGAATTGGGT 
			TTAAACATGAGC 
			CCCAAGAAGAA</td>
			<td rowspan="2">pCasRx</td>
		</tr>
		<tr>
			<td>1050E.C4</td>
			<td>CAATTGATTTGT 
			TATTTTAAAAAC 
			GATTCATTCTA 
			GCTAGCTTAAG 
			CGTAATCTGGA 
			ACA</td>
		</tr>
		<tr>
			<td rowspan="4">OA-1050S</td>
			<td rowspan="2">UASt promoter</td>
			<td>1041.C9</td>
			<td>GCGGGTTCTC 
			GACGGTCACG 
			GCGGGCATGT 
			CGACGCGGCC 
			GCAACCAACAA 
			CACTAGTAG</td>
			<td rowspan="2">pJFRC81</td>
		</tr>
		<tr>
			<td>1041.C11</td>
			<td>CTGGCCTCCA 
			CCTTTCTCTTC 
			TTCTTGGGGCT 
			CATGTTTAAAC 
			CCAATTCCCTA 
			TTCAGA</td>
		</tr>
		<tr>
			<td rowspan="2">dCasRx</td>
			<td>1050L.C1</td>
			<td>AATACAAGAAG 
			AGAACTCTGAAT 
			AGGGAATTGGG 
			TTTAAACATGAG 
			CCCCAAGAAGAA</td>
			<td rowspan="2">pdCasRx</td>
		</tr>
		<tr>
			<td>1050E.C4</td>
			<td>CAATTGATTTGT 
			TATTTTAAAAAC 
			GATTCATTCTAG 
			CTAGCTTAAGCG 
			TAATCTGGAACA</td>
		</tr>
		<tr>
			<td rowspan="2">OA-1043</td>
			<td rowspan="2">U6:3 promoter</td>
			<td>1043.C1</td>
			<td>GGGAATTGGGA 
			ATTGGGCAATAT 
			TTAAATGGCGGC 
			GCGCCGAATTCT 
			TTTTTGCTCACCT</td>
			<td rowspan="2">Addgene plasmid #164586</td>
		</tr>
		<tr>
			<td>1043.C23</td>
			<td>ACACTAGTGGAT 
			CTCTAGAGGTAC 
			CGTTGCGGCCG 
			CAAAAAAGTTGT 
			AATAGCCCCTCA 
			AAACTGGACCTT 
			CCACAACTGCAG 
			CCGACGTTAAAT 
			TGAAA</td>
		</tr>
		<tr>
			<td rowspan="6">OA-1052B</td>
			<td rowspan="2">Ubiq promoter</td>
			<td>1052B.C1</td>
			<td>GGGAATTGGGCA 
			ATATTTAAATGGC 
			GGCTGCAGCGC 
			GCAGATCGCCGAT</td>
			<td rowspan="2">Addgene plasmid #112686</td>
		</tr>
		<tr>
			<td>1052B.C2</td>
			<td>TTTCTTTATGTTT 
			TTGGCGTCTTCC 
			ATCCTAGGTCTG 
			CGGGTCAAAATA 
			GAGATG</td>
		</tr>
		<tr>
			<td rowspan="2">T2A-eGFP</td>
			<td>908A1</td>
			<td>ATAAAGGCCAAG 
			AAGGGCGGAAA 
			GATCGCCGTGG 
			AGGGCAGAGGA 
			AGTCTTCTAACAT 
			GC</td>
			<td rowspan="2">Addgene plasmid #112686</td>
		</tr>
		<tr>
			<td>908A2</td>
			<td>TTGTTATTTTAAAA 
			ACGATTCATTCTA 
			GGCGATCGCTTA 
			CTTGTACAGCTC 
			GTCCATGCC</td>
		</tr>
		<tr>
			<td rowspan="2">Reversed Ubiq promoter</td>
			<td>908A3</td>
			<td>ACCGTGACCTAC 
			ATCGTCGACACTA 
			GTGGATCTCTAGA 
			CGCGCAGATCGC 
			CGATG</td>
			<td rowspan="2">Addgene plasmid #112686</td>
		</tr>
		<tr>
			<td>908A4</td>
			<td>GGATCATAAACTT 
			TCGAAGTCATGC 
			GGCCGCTCTGCG 
			GGTCAAAATAGAG 
			ATGT</td>
		</tr>
	</tbody>
</table>
Table 1: List of molecular construct and primers used in this study. This list includes all constructs (both the ID and description) and each construct&#39;s associated primers (both the ID and sequences (5&#39; to 3&#39;)) and templates used.

Watch this Scientific Journal Video about Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx at JoVE.com

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx

This article outlines a detailed protocol for using the RNA-targeting Cas13D enzyme (RfxCas13D) in flies.

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx

CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with ...

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RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...