J Genomics 2020; 8:30-36. doi:10.7150/jgen.43928 This volume Cite

Research Paper

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

Aditi Kulkarni, Wanqin Yu, Alex S. Moon, Ashmita Pandey, Kathryn A. Hanley, Jiannong Xu

Department of Biology, New Mexico State University, PO Box 30001 MSC 3AF, Las Cruces NM, 88003, USA

Citation:
Kulkarni A, Yu W, Moon AS, Pandey A, Hanley KA, Xu J. Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes. J Genomics 2020; 8:30-36. doi:10.7150/jgen.43928. https://www.jgenomics.com/v08p0030.htm
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Abstract

Graphic 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.

Keywords: CRISPR-Cas13a, RNA interference, Anopheles gambiae, Aedes aegypti, gene silencing, CRISPRi


Citation styles

APA
Kulkarni, A., Yu, W., Moon, A.S., Pandey, A., Hanley, K.A., Xu, J. (2020). Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes. Journal of Genomics, 8, 30-36. https://doi.org/10.7150/jgen.43928.

ACS
Kulkarni, A.; Yu, W.; Moon, A.S.; Pandey, A.; Hanley, K.A.; Xu, J. Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes. J. Genomics 2020, 8, 30-36. DOI: 10.7150/jgen.43928.

NLM
Kulkarni A, Yu W, Moon AS, Pandey A, Hanley KA, Xu J. Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes. J Genomics 2020; 8:30-36. doi:10.7150/jgen.43928. https://www.jgenomics.com/v08p0030.htm

CSE
Kulkarni A, Yu W, Moon AS, Pandey A, Hanley KA, Xu J. 2020. Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes. J Genomics. 8:30-36.

This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
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