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Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes
Aditi Kulkarni1, Wanqin Yu1, Alex S Moon1
1Department of Biology, New Mexico State University, PO Box 30001 MSC 3AF, Las Cruces NM, 88003, USA.
Abstract:
In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.
Insights
Researchers developed a CRISPR interference tool using Cas13a to silence specific genes in mosquitoes, offering a new method for gene knockdown with no observed off-target effects.
Area of Science:
- Molecular Biology
- Genetics
- Vector Control
Background:
- CRISPR-Cas systems offer precise gene editing capabilities.
- Cas13a is an RNA-guided RNA nuclease targeting single-stranded RNA.
- Gene silencing in mosquitoes is crucial for vector control research.
Purpose of the Study:
- To develop and evaluate a Cas13a-mediated CRISPR interference (CRISPRi) tool for gene silencing in mosquitoes.
- To assess the efficacy and specificity of this tool in different mosquito species.
Main Methods:
- Development of a Cas13a-expressing plasmid for intrathoracic injection into mosquitoes.
- Synthesis of target-specific CRISPR RNA (crRNA) in vitro.
- Delivery of Cas13a and crRNA to Anopheles gambiae and Aedes aegypti.
- Analysis of gene silencing effects on target genes (vitellogenin, COPI subunits) and organismal phenotypes.
Main Results:
- Cas13a transcripts were detected for at least 10 days post-injection.
- Successful silencing of the vitellogenin gene in Anopheles gambiae led to reduced egg production.
- Silencing of COPI gene subunits in Aedes aegypti resulted in mortality and midgut fragility.
- Simultaneous gene silencing was achieved using a crRNA cocktail.
- No significant collateral cleavage of non-target transcripts was observed.
Conclusions:
- Cas13a-mediated CRISPR interference is a viable and programmable tool for gene knockdown in mosquitoes.
- This method provides an alternative to traditional RNA interference (RNAi) techniques like dsRNA or siRNA.
- The CRISPRi system demonstrates high specificity and potential for applications in mosquito research and control.
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