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• W3048341121 abstract "•CRISPR-RfxCas13d knocks down maternal and zygotic mRNA in zebrafish embryos•Both RfxCas13d protein and mRNA can be used to recapitulate developmental phenotypes•CRISPR-RfxCas13d is an efficient tool to interrogate embryonic gene function•CRISPR-RfxCas13d is also functional in medaka, killifish, and mouse embryos Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos. Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos. The experimental dissection of gene function has been radically transformed with the advent of DNA engineering technologies, such as TALEN and CRISPR-Cas9 systems (Hsu et al., 2014Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (3100) Google Scholar). These tools allow researchers to link genotype to cellular phenotype through the generation of permanent changes to the genome. Complementary “knockdown” approaches such as RNA interference have also proven to be invaluable tools to interrogate gene function. However, in aquatic vertebrate organisms, such as zebrafish (Danio rerio), other teleost fish, and Xenopus (Chen et al., 2017Chen G.R. Sive H. Bartel D.P. A seed mismatch enhances Argonaute2-catalyzed cleavage and partially rescues severely impaired cleavage found in fish.Mol. Cell. 2017; 68: 1095-1107.e5Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar; Lund et al., 2011Lund E. Sheets M.D. Imboden S.B. Dahlberg J.E. Limiting Ago protein restricts RNAi and microRNA biogenesis during early development in Xenopus laevis.Genes Dev. 2011; 25: 1121-1131Crossref PubMed Scopus (71) Google Scholar), attempts to establish efficient RNAi technologies have largely failed (Chen et al., 2017Chen G.R. Sive H. Bartel D.P. A seed mismatch enhances Argonaute2-catalyzed cleavage and partially rescues severely impaired cleavage found in fish.Mol. Cell. 2017; 68: 1095-1107.e5Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar; Kelly and Hurlstone, 2011Kelly A. Hurlstone A.F. The use of RNAi technologies for gene knockdown in zebrafish.Brief. Funct. Genomics. 2011; 10: 189-196Crossref PubMed Scopus (30) Google Scholar; Lund et al., 2011Lund E. Sheets M.D. Imboden S.B. Dahlberg J.E. Limiting Ago protein restricts RNAi and microRNA biogenesis during early development in Xenopus laevis.Genes Dev. 2011; 25: 1121-1131Crossref PubMed Scopus (71) Google Scholar). In their place, morpholinos (MOs), and more recently, antisense oligonucleotides (ASOs), have been used to perturb RNA activity (Pauli et al., 2015Pauli A. Montague T.G. Lennox K.A. Behlke M.A. Schier A.F. Antisense oligonucleotide-mediated transcript knockdown in zebrafish.PLoS One. 2015; 10: e0139504Crossref PubMed Scopus (17) Google Scholar; Stainier et al., 2017Stainier D.Y.R. Raz E. Lawson N.D. Ekker S.C. Burdine R.D. Eisen J.S. Ingham P.W. Schulte-Merker S. Yelon D. Weinstein B.M. et al.Guidelines for morpholino use in zebrafish.PLoS Genet. 2017; 13: e1007000Crossref PubMed Scopus (168) Google Scholar). While an ASO-based approach has not been followed up since its implementation in zebrafish (Pauli et al., 2015Pauli A. Montague T.G. Lennox K.A. Behlke M.A. Schier A.F. Antisense oligonucleotide-mediated transcript knockdown in zebrafish.PLoS One. 2015; 10: e0139504Crossref PubMed Scopus (17) Google Scholar), MOs have been widely implemented for over two decades. MOs are nucleic acid-analog antisense oligomers that disrupt RNA function or output by blocking translation or splicing (Stainier et al., 2017Stainier D.Y.R. Raz E. Lawson N.D. Ekker S.C. Burdine R.D. Eisen J.S. Ingham P.W. Schulte-Merker S. Yelon D. Weinstein B.M. et al.Guidelines for morpholino use in zebrafish.PLoS Genet. 2017; 13: e1007000Crossref PubMed Scopus (168) Google Scholar). However, the utility of MOs has recently been called into question due to observed toxicity and off-target effects (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar; Joris et al., 2017Joris M. Schloesser M. Baurain D. Hanikenne M. Muller M. Motte P. Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.Nucleic Acids Res. 2017; 45: 9547-9557Crossref PubMed Scopus (16) Google Scholar; Kok et al., 2015Kok F.O. Shin M. Ni C.W. Gupta A. Grosse A.S. van Impel A. Kirchmaier B.C. Peterson-Maduro J. Kourkoulis G. Male I. et al.Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.Dev. Cell. 2015; 32: 97-108Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar; Lai et al., 2019Lai J.K.H. Gagalova K.K. Kuenne C. El-Brolosy M.A. Stainier D.Y.R. Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.Dev. Biol. 2019; 454: 21-28Crossref PubMed Scopus (14) Google Scholar; Robu et al., 2007Robu M.E. Larson J.D. Nasevicius A. Beiraghi S. Brenner C. Farber S.A. Ekker S.C. p53 activation by knockdown technologies.PLoS Genet. 2007; 3: e78Crossref PubMed Scopus (794) Google Scholar; Schulte-Merker and Stainier, 2014Schulte-Merker S. Stainier D.Y. Out with the old, in with the new: reassessing morpholino knockdowns in light of genome editing technology.Development. 2014; 141: 3103-3104Crossref PubMed Scopus (120) Google Scholar). In several instances, discordant phenotypes have been observed between MO-treated animals and loss-of-function mutant animals, and more strikingly, additional phenotypes have been observed upon MO treatment in loss-of-function mutant backgrounds, indicating cellular effects unrelated to targeted RNA knockdown (Joris et al., 2017Joris M. Schloesser M. Baurain D. Hanikenne M. Muller M. Motte P. Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.Nucleic Acids Res. 2017; 45: 9547-9557Crossref PubMed Scopus (16) Google Scholar; Kok et al., 2015Kok F.O. Shin M. Ni C.W. Gupta A. Grosse A.S. van Impel A. Kirchmaier B.C. Peterson-Maduro J. Kourkoulis G. Male I. et al.Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.Dev. Cell. 2015; 32: 97-108Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). More recently, studies have shown that some MOs can trigger an innate immunity response, activation of interferon, and cellular stress response pathways, as well as off-targeting through mis-splicing in zebrafish and Xenopus (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar; Joris et al., 2017Joris M. Schloesser M. Baurain D. Hanikenne M. Muller M. Motte P. Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.Nucleic Acids Res. 2017; 45: 9547-9557Crossref PubMed Scopus (16) Google Scholar; Lai et al., 2019Lai J.K.H. Gagalova K.K. Kuenne C. El-Brolosy M.A. Stainier D.Y.R. Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.Dev. Biol. 2019; 454: 21-28Crossref PubMed Scopus (14) Google Scholar; Robu et al., 2007Robu M.E. Larson J.D. Nasevicius A. Beiraghi S. Brenner C. Farber S.A. Ekker S.C. p53 activation by knockdown technologies.PLoS Genet. 2007; 3: e78Crossref PubMed Scopus (794) Google Scholar), reinforcing the need for established controls to ensure the fidelity of MO-induced phenotypes (Stainier et al., 2017Stainier D.Y.R. Raz E. Lawson N.D. Ekker S.C. Burdine R.D. Eisen J.S. Ingham P.W. Schulte-Merker S. Yelon D. Weinstein B.M. et al.Guidelines for morpholino use in zebrafish.PLoS Genet. 2017; 13: e1007000Crossref PubMed Scopus (168) Google Scholar). These concerns, as well as the high expense of MOs, tedious methods for validating efficacy and fidelity (i.e., assessment of protein output or ribosome occupancy) (Lee et al., 2013Lee M.T. Bonneau A.R. Takacs C.M. Bazzini A.A. DiVito K.R. Fleming E.S. Giraldez A.J. Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition.Nature. 2013; 503: 360-364Crossref PubMed Scopus (255) Google Scholar), and the inability to implement in a tissue-specific or temporal manner have limited their use in systematic approaches to studying gene function in teleost and amphibian embryos. RNA “knockdown” approaches hold many advantages compared with DNA mutagenesis (e.g., Cas9). The ability to directly disrupt gene activity sidesteps the need for laborious multi-generation genotype screening to establish permanent genetic strains. Likewise, knockdown strategies allow for the study of in vivo maternal effects without the need for homozygous mutant mothers (Ciruna et al., 2002Ciruna B. Weidinger G. Knaut H. Thisse B. Thisse C. Raz E. Schier A.F. Production of maternal-zygotic mutant zebrafish by germ-line replacement.Proc. Natl. Acad. Sci. USA. 2002; 99: 14919-14924Crossref PubMed Scopus (162) Google Scholar; Moreno-Mateos et al., 2015Moreno-Mateos M.A. Vejnar C.E. Beaudoin J.D. Fernandez J.P. Mis E.K. Khokha M.K. Giraldez A.J. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.Nat. Methods. 2015; 12: 982-988Crossref PubMed Scopus (517) Google Scholar), which for many critically important genes are either unviable or infertile (Abrams and Mullins, 2009Abrams E.W. Mullins M.C. Early zebrafish development: it's in the maternal genes.Curr. Opin. Genet. Dev. 2009; 19: 396-403Crossref PubMed Scopus (100) Google Scholar; Ciruna et al., 2002Ciruna B. Weidinger G. Knaut H. Thisse B. Thisse C. Raz E. Schier A.F. Production of maternal-zygotic mutant zebrafish by germ-line replacement.Proc. Natl. Acad. Sci. USA. 2002; 99: 14919-14924Crossref PubMed Scopus (162) Google Scholar). Furthermore, by directly manipulating RNA activity in a relatively tunable manner, researchers can study how subtle changes in transcript levels impact biological processes. In fact, by reducing but not completely removing gene activity, researchers can uncover phenotypes that may otherwise remain hidden due to loss-of-function lethality (Smith et al., 2017Smith I. Greenside P.G. Natoli T. Lahr D.L. Wadden D. Tirosh I. Narayan R. Root D.E. Golub T.R. Subramanian A. Doench J.G. Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the connectivity map.PLoS Biol. 2017; 15: e2003213Crossref PubMed Scopus (78) Google Scholar). Finally, therapeutic approaches that target RNA rather than DNA can be advantageous in providing temporary but effective modifications without the prospect of permanent heritable changes (Cox et al., 2017Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027Crossref PubMed Scopus (604) Google Scholar). Cas13 is a class 2 type VI CRISPR-Cas RNA endonuclease, which has recently been employed to induce both the cleavage and subsequent degradation of RNA in fission yeast, plants, and mammalian cell lines (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (642) Google Scholar; Aman et al., 2018Aman R. Ali Z. Butt H. Mahas A. Aljedaani F. Khan M.Z. Ding S. Mahfouz M. RNA virus interference via CRISPR/Cas13a system in plants.Genome Biol. 2018; 19: 1Crossref PubMed Scopus (150) Google Scholar; Cox et al., 2017Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027Crossref PubMed Scopus (604) Google Scholar; Jing et al., 2018Jing X. Xie B. Chen L. Zhang N. Jiang Y. Qin H. Wang H. Hao P. Yang S. Li X. Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing.Nucleic Acids Res. 2018; 46: e90Crossref PubMed Scopus (21) Google Scholar; Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676.e14Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). This method has been shown to be more effective and specific than RNAi in mammalian cells (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (642) Google Scholar; Cox et al., 2017Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027Crossref PubMed Scopus (604) Google Scholar; Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676.e14Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). However, to date, no systematic study of the potential of Cas13 has been carried out in an animal model system. Here, we show that CRISPR-RfxCas13d (CasRx), but not Psp/PguCas13b or LwaCas13a, is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed as well as ectopic or maternally provided transcripts are efficiently targeted, resulting in an average of more than 76% decrease in transcript levels and the recapitulation of well-known maternal and/or zygotic embryonic phenotypes. Importantly, through whole transcriptome sequencing, we observed specific knockdown of target mRNA. In addition, we show that the use of purified Cas13d protein increases maternal phenotype penetrance, while preserving the specificity observed with Cas13d mRNA. Finally, we successfully implement CRISPR-Cas13d system in other research organisms such as medaka (Oryzias latipes), killifish (Nothobranchius furzeri), and mouse (Mus musculus) embryos, demonstrating its applicability across a range of aquatic and terrestrial animal models. Our results establish CRISPR-Cas13d as an efficient and straightforward method for the systematic, tractable, and unambiguous study of gene function in vivo during embryogenesis across a range of animal species. Gene knockdown approaches such as RNAi serve an invaluable research tools toward elucidating gene function. However, in aquatic vertebrate organisms, such as zebrafish and other teleost fish, RNAi has failed to develop as an efficient and systematic tool. Instead, MOs have been used to disrupt gene function by blocking translation or preventing RNA splicing. However, MOs can induce nonspecific effects that are separate from target RNA perturbation (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar; Joris et al., 2017Joris M. Schloesser M. Baurain D. Hanikenne M. Muller M. Motte P. Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.Nucleic Acids Res. 2017; 45: 9547-9557Crossref PubMed Scopus (16) Google Scholar; Lai et al., 2019Lai J.K.H. Gagalova K.K. Kuenne C. El-Brolosy M.A. Stainier D.Y.R. Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.Dev. Biol. 2019; 454: 21-28Crossref PubMed Scopus (14) Google Scholar; Robu et al., 2007Robu M.E. Larson J.D. Nasevicius A. Beiraghi S. Brenner C. Farber S.A. Ekker S.C. p53 activation by knockdown technologies.PLoS Genet. 2007; 3: e78Crossref PubMed Scopus (794) Google Scholar). Further, researchers have found that MO-based phenotypes can differ from those obtained by genetic mutation (Kok et al., 2015Kok F.O. Shin M. Ni C.W. Gupta A. Grosse A.S. van Impel A. Kirchmaier B.C. Peterson-Maduro J. Kourkoulis G. Male I. et al.Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.Dev. Cell. 2015; 32: 97-108Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). Although these differences may be explained, in part, by genetic compensation (El-Brolosy et al., 2019El-Brolosy M.A. Kontarakis Z. Rossi A. Kuenne C. Günther S. Fukuda N. Kikhi K. Boezio G.L.M. Takacs C.M. Lai S.L. et al.Genetic compensation triggered by mutant mRNA degradation.Nature. 2019; 568: 193-197Crossref PubMed Scopus (331) Google Scholar; Ma et al., 2019Ma Z. Zhu P. Shi H. Guo L. Zhang Q. Chen Y. Chen S. Zhang Z. Peng J. Chen J. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components.Nature. 2019; 568: 259-263Crossref PubMed Scopus (166) Google Scholar; Rossi et al., 2015Rossi A. Kontarakis Z. Gerri C. Nolte H. Hölper S. Krüger M. Stainier D.Y. Genetic compensation induced by deleterious mutations but not gene knockdowns.Nature. 2015; 524: 230-233Crossref PubMed Scopus (688) Google Scholar), the use of MOs requires several proper controls to ensure accuracy (Stainier et al., 2017Stainier D.Y.R. Raz E. Lawson N.D. Ekker S.C. Burdine R.D. Eisen J.S. Ingham P.W. Schulte-Merker S. Yelon D. Weinstein B.M. et al.Guidelines for morpholino use in zebrafish.PLoS Genet. 2017; 13: e1007000Crossref PubMed Scopus (168) Google Scholar). Based on the limitations and caveats of current knockdown technologies in teleosts, we set out to develop and optimize a CRISPR-Cas13-based method to disrupt gene function. We had two goals: (1) develop an approach to interrogate maternally provided developmental programs in the early embryo and (2) develop a technique that could be used in a systematic and cost-efficient manner. The study of early development in animal embryos, which is driven by thousands of mRNAs that are maternally provided (Lee et al., 2014Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition.Annu. Rev. Cell Dev. Biol. 2014; 30: 581-613Crossref PubMed Scopus (281) Google Scholar), has traditionally required elaborate genetic schemes to remove maternal activity (Ciruna et al., 2002Ciruna B. Weidinger G. Knaut H. Thisse B. Thisse C. Raz E. Schier A.F. Production of maternal-zygotic mutant zebrafish by germ-line replacement.Proc. Natl. Acad. Sci. USA. 2002; 99: 14919-14924Crossref PubMed Scopus (162) Google Scholar). Herein, we find that, among various Cas13 variants tested, RfxCas13d efficiently disrupts maternal and zygotic (MZ) gene function without embryonic toxicity, and with specificity, in zebrafish embryos. Notably, Cas13d protein injection accelerates maternal RNA depletion and consequently provides more penetrant phenotypes. Through successful application in medaka, killifish, and mouse embryos, we find that CRISPR-RfxCas13d provides a fast, robust, and cost-effective technology to systematically disrupt gene function in vertebrates. We envision the application of this method to the high-throughput study of gene expression in the early embryo, as well as in combination with inducible and tissue-specific methodologies. To assess the potential toxic effects of different Cas13 proteins successfully used in mammalian cells (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (642) Google Scholar; Cox et al., 2017Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027Crossref PubMed Scopus (604) Google Scholar; Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676.e14Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), we constructed expression vectors to generate comparable mRNA encoding four different Cas13 variants (RfxCas13d, PguCas13b, PspCas13b, or LwaCas13a-GFP). The mRNA for each Cas13 variant was injected individually into one-cell stage zebrafish embryos (Figure 1A). Protein expression in vivo was confirmed for all Cas13 variants tested (Figure S1A) except LwaCas13a-GFP likely due to a low stability of LwaCas13 mRNA as reported in mammalian cells (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (642) Google Scholar) (Figure S1B). Although PguCas13b and PspCas13b negatively impacted embryonic development (Figures 1B and S1C), RfxCas13d displayed no toxic effects (up to 300 pg of mRNA per embryo; Figure S1D). Hence, we limited subsequent analyses of endogenous mRNA abrogation to RfxCas13d. First, to test whether RfxCas13d could trigger the degradation of specific mRNAs in zebrafish embryos, we designed and generated six guide RNAs (gRNAs) complementary to different sequences within the coding sequence (CDS) of the tbxta mRNA pooled into 2 sets of gRNAs (Figures 1C, S1E, and S1F). Notochord formation is compromised in tbxta loss-of-function mutant embryos as well as a lack of posterior structures (no-tail) (Halpern et al., 1993Halpern M.E. Ho R.K. Walker C. Kimmel C.B. Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation.Cell. 1993; 75: 99-111Abstract Full Text PDF PubMed Scopus (461) Google Scholar; Schulte-Merker et al., 1994Schulte-Merker S. Hammerschmidt M. Beuchle D. Cho K.W. De Robertis E.M. Nüsslein-Volhard C. Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos.Development. 1994; 120: 843-852Crossref PubMed Google Scholar). Co-injection of RfxCas13d mRNA (mCas13d) with different sets of tbxta gRNAs (Figure 1C) recapitulated the no-tail phenotype at 30 h post-injection (hpi) (Figures 1D, 1E, S1G, and S1H) (Strähle et al., 1996Strähle U. Blader P. Ingham P.W. Expression of axial and sonic hedgehog in wildtype and midline defective zebrafish embryos.Int. J. Dev. Biol. 1996; 40: 929-940PubMed Google Scholar) and persist for at least 4 days post-injection (Figure S1I). Importantly, the no-tail phenotype was only observed when tbxta gRNAs and mCas13d were co-injected (i.e., no phenotype was observed when either gRNAs or mCas13d were injected singly), and its penetrance correlated with mCas13d dosage (Figure 1E). Additionally, chemically synthesized (Synt) gRNAs generated a similar number of no-tail embryos but with a slight increase in phenotype penetrance (Figure S1J). Further, co-injection of mCas13d with gRNAs targeting dnd1, a gene controlling an unrelated developmental process (germ cell development and survival) (Weidinger et al., 2003Weidinger G. Stebler J. Slanchev K. Dumstrei K. Wise C. Lovell-Badge R. Thisse C. Thisse B. Raz E. Dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival.Curr. Biol. 2003; 13: 1429-1434Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), did not produce a no-tail phenotype (Figure 1F). In addition, the CRISPR-RfxCas13d system triggered tbxta mRNA degradation by at least 6 hpi (Figure 1G) (p < 0.003, t test). While the addition of nuclear localization sites (NLSs) increased RfxCas13d activity in mammalian cells (Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676.e14Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), we observed that incorporation of an NLS decreased phenotypic penetrance in zebrafish embryos (Figure S1K) (p < 3.4e−9, chi-square test). Finally, to confirm the specificity of the phenotype, we designed a new set of three gRNAs targeting the tbxta 3′UTR, which also induced the no-tail phenotype (Figure 1F). This phenotype was partially rescued (p < 2.07e−3, chi-square test) by injection of the cognate mRNA (lacking endogenous 3′UTR), but not by injection of mRNA encoding GFP or the antisense tbxta coding region (p < 0.02 and 0.29, respectively; chi-square test) (Figures 1F and S1L). To test the ability of targeting of individual gRNAs, we injected Cas13d mRNA together with nine individual and different gRNAs targeting tbxta mRNA and observed that all of them recapitulated the no-tail phenotype with variable penetrance (Figure S1M). All together, these results establish CRISPR-RfxCas13d as an efficient tool to trigger endogenous mRNA knockdown during zebrafish embryogenesis. To further assess the efficacy of the CRISPR-RfxCas13d system to impair mRNA activity, two sets of three gRNAs each, specific to the CDS of either red fluorescent protein (RFP) or nano-luciferase, were individually injected together with their corresponding controls (gfp and firefly mRNAs, respectively) along with mCas13d into zebrafish embryos (Figures 2A and 2B ). Reporter activity (Figures 2C and 2D), as well as the respective mRNA levels (Figures 2E and 2F), were significantly reduced in zebrafish embryos after 6 hpi when compared with embryos injected with control conditions (Figures 2C–2F). Moreover, mCas13d coupled with individual gRNAs was sufficient to reduce RFP fluorescence intensity (Figures S2A and S2B). Notably, fluorescence intensity of GFP and firefly, respectively, did not decrease appreciably in all conditions tested (Figures 2C and S2A–S2C), and only targeted mRNAs (rfp and nano-luciferase) displayed reduced mRNA levels (Figures 2C–2F, S2A, and S2B), suggesting that the CRISPR-RfxCas13d system is specific. To address whether the CRISPR-RfxCas13d system could be used to target maternally deposited mRNAs, zebrafish embryos derived from a transgenic mother that expresses rfp at very high levels (i.e., among the top 100 most highly expressed maternal mRNAs) were injected with mCas13d, gfp mRNA, plus three gRNAs targeting rfp (Figure 2G). Fluorescence intensity was visibly reduced after 24 hpi when compared with embryos injected with mCas13d or gRNAs alone, or by co-injection of mCas13d with unrelated gRNAs (Figure 2H). Interestingly, the reduced level of RFP persisted for the first 3.5 days and partially recovered after 5 days (Figures S2D and S2E, respectively). Furthermore, rfp mRNA levels displayed an approximately 4.4-fold reduction (77.3%; p adjusted < 7.64e−6) compared with embryos injected with mCas13d alone after 6 hpi by RNA sequencing (RNA-seq) (Figure 2I) and qRT-PCR (Figure S2F). Moreover, RNA-seq analysis displayed similar transcript levels in zebrafish embryos injected with mCas13d alone or uninjected embryos (Figures 2I and S2G). Importantly, within the co-injected embryos with mCas13d and gRNAs targeting rfp mRNA, rfp was the most significantly downregulated transcript (with >4-fold change) (Figures 2I and S2G). Together, these results indicate that the CRISPR-RfxCas13d system can functionally disrupt the activity of maternally provided mRNAs in a specific manne" @default.
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• W3048341121 date "2020-09-01" @default.
• W3048341121 modified "2023-10-15" @default.
• W3048341121 title "CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos" @default.
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• W3048341121 doi "https://doi.org/10.1016/j.devcel.2020.07.013" @default.
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