A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila
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A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila Nhan Huynh, Noah Depner, Raegan Larson and Kirst King-Jones* * Correspondence: kirst.king-jones@ ualberta.ca Department of Biological Sciences, University of Alberta, G-504 Biological Sciences Bldg., Edmonton, Alberta T6G 2E9, Canada
Abstract Advances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level. Keywords: CRISPR, Cas13, CasRX, Drosophila, RNA manipulation, crRNA design
Background Most bacterial and archaeal genomes harbor clustered regularly interspaced short palindromic repeats (CRISPR) and encode CRISPR-associated proteins (Cas) as a defense system against bacteriophages and other invading nucleic acids [1–3]. The immune response of all CRISPR/Cas systems characterized to date includes three steps: (i) adaptation and spacer acquisition, where a piece of the invading genome is incorporated into the CRISPR array, (ii) the expression of mature CRISPR-RNAs (gRNAs) from the processed CRISPR array, and (iii) interference, where Cas enzymes are guided by the gRNAs to the corresponding region of the invading genome for cleavage and degradation [4, 5]. The CRISPR/Cas class II systems use a single, multidomain Cas effector protein [6]. Because of its simplicity, the single multidomain effector found in class II organisms is used in current CRISPR methods. Class II type II CRISPR Cas9 was one of the first Cas proteins studied in detail, which led to its widespread use for genomic engineering (Fig. 1a) [6–11]. Currently, CRISPR/Cas9 approaches allow scientists to precisely alter gene function via (i) classic CRISPR to introduce short INDELs, (ii) HRbased CRISPR for homology-based gene replacements or deletions, (iii) somatic CRIS PR for conditional gene disruption, (iv) CRISPRi, (i = interference) to interfere with gene transcription, and (v) CRISPRa (a = activation) to upregulate gene activity. Studies have shown that it is possible to conditionally target genes of interest by exerting © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material i
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