Name: determining the role of maternally-expressed genes in early development with maternal crispants
Uploaded: 2021-12-21T14:00:00
Description: Watch this Scientific Journal Video about Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants at JoVE.com

We thank past and current Pelegri lab animal husbandry staff members for their care of the aquatic facility. We are also grateful for the comments and insight on the manuscript by Ryan Trevena and Diane Hanson. Funding was provided by NIH grant to F.P. (GM065303)

In studies leading to the development of this protocol, all zebrafish housing and experiments were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (IACUC-M005268-R2).
1. Synthesis of Guide RNAs
NOTE: Zygotic crispants have been created using a single guide RNA or multiplexing multiple guide RNAs to a single target5,6,7</s...

<ol>
	<li>Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+factors+in+zebrafish+development.">Maternal factors in zebrafish development.</a> Developmental Dynamics. 228 (3), 535-554 (2003).</li><li>Campbell, P. D., Heim, A. E., Smith, M. Z., Marlow, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-1+interacts+with+Bucky+ball+to+form+germ+cells+and+is+required+to+pattern+the+zebrafish+body+axis.">Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis.</a> Development. 142 (17), 2996-3008 (2015).</li><li>Eno, C., Solanki, B., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=aura+(mid1ip1l)+regulates+the+cytoskeleton+at+the+zebrafish+egg-to-embryo+transition.">aura (mid1ip1l) regulates the cytoskeleton at the zebrafish egg-to-embryo transition.</a> Development. 143 (9), 1585-1599 (2016).</li><li>He, W. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-specific+maternal+Slbp2+is+required+for+replication-dependent+histone+storage+and+early+nuclear+cleavage+in+zebrafish+oogenesis+and+embryogenesis.">Oocyte-specific maternal Slbp2 is required for replication-dependent histone storage and early nuclear cleavage in zebrafish oogenesis and embryogenesis.</a> RNA. 24 (12), 1738-1748 (2018).</li><li>Burger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mutagenesis+with+solubilized+CRISPR-Cas9+ribonucleoprotein+complexes.">Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.</a> Development. 143 (11), 2025-2037 (2016).</li><li>Jao, L. -. E., Wente, S. R., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+multiplex+biallelic+zebrafish+genome+editing+using+a+CRISPR+nuclease+system.">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (34), 13904-13909 (2013).</li><li>Shah, A. N., Davey, C. F., Whitebirch, A. C., Miller, A. C., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+reverse+genetic+screening+using+CRISPR+in+zebrafish.">Rapid reverse genetic screening using CRISPR in zebrafish.</a> Nature Methods. 12 (6), 535-540 (2015).</li><li>Shankaran, S. S., Dahlem, T. J., Bisgrove, B. W., Yost, H. J., Tristani-Firouzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-directed+gene+editing+for+the+generation+of+loss-of-function+mutants+in+high-throughput+zebrafish+F0+screens.">CRISPR/Cas9-directed gene editing for the generation of loss-of-function mutants in high-throughput zebrafish F0 screens.</a> Current Protocols in Molecular Biology. 119 (1), 1-22 (2017).</li><li>Trubiroha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+CRISPR/Cas-based+mutagenesis+assay+in+zebrafish+for+identification+of+genes+involved+in+thyroid+morphogenesis+and+function.">A Rapid CRISPR/Cas-based mutagenesis assay in zebrafish for identification of genes involved in thyroid morphogenesis and function.</a> Scientific Reports. 8 (1), 5647 (2018).</li><li>Wu, R. S., Lam, I. I., Clay, H., Duong, D. N., Deo, R. C., Coughlin, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+method+for+directed+gene+knockout+for+screening+in+G0+zebrafish.">A Rapid method for directed gene knockout for screening in G0 zebrafish.</a> Developmental Cell. 46 (1), 112-125 (2018).</li><li>Moravec, C. E., Voit, G. C., Otterlee, J., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+maternal-effect+genes+in+zebrafish+using+maternal+crispants.">Identification of maternal-effect genes in zebrafish using maternal crispants.</a> Development. 148 (19), 199536 (2021).</li><li>Braat, A. K., Zandbergen, T., van de Water, S., Goos, H. J., Zivkovic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+primordial+germ+cells:+morphology+and+early+distribution+of+vasa+RNA.">Characterization of zebrafish primordial germ cells: morphology and early distribution of vasa RNA.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 216 (2), 153-167 (1999).</li><li>Eno, C., Hansen, C. L., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation,+segregation,+and+dispersal+of+homotypic+germ+plasm+RNPs+in+the+early+zebrafish+embryo.">Aggregation, segregation, and dispersal of homotypic germ plasm RNPs in the early zebrafish embryo.</a> Developmental Dynamics. 248 (4), 306-318 (2019).</li><li>Knaut, H., Steinbeisser, H., Schwarz, H., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolutionary+conserved+region+in+the+vasa+3'UTR+targets+RNA+translation+to+the+germ+cells+in+the+zebrafish.">An evolutionary conserved region in the vasa 3'UTR targets RNA translation to the germ cells in the zebrafish.</a> Current biology: CB. 12 (6), 454-466 (2002).</li><li>Yoon, C., Kawakami, K., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+vasa+homologue+RNA+is+localized+to+the+cleavage+planes+of+2-+and+4-cell-stage+embryos+and+is+expressed+in+the+primordial+germ+cells.">Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells.</a> Development. 124 (16), 3157-3165 (1997).</li><li>Gagnon, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+mutagenesis+by+Cas9+protein-mediated+oligonucleotide+insertion+and+large-scale+assessment+of+single-guide+RNAs.">Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.</a> PLoS ONE. 9 (5), 98186 (2014).</li><li>Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B., Valen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHOPCHOP+v2:+a+web+tool+for+the+next+generation+of+CRISPR+genome+engineering.">CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering.</a> Nucleic Acids Research. 44, 272-276 (2016).</li><li>Labun, K., Montague, T. G., Krause, M., Torres Cleuren, Y. N., Tjeldnes, H., Valen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHOPCHOP+v3:+expanding+the+CRISPR+web+toolbox+beyond+genome+editing.">CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing.</a> Nucleic Acids Research. 47, 171-174 (2019).</li><li>Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M., Valen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHOPCHOP:+a+CRISPR/Cas9+and+TALEN+web+tool+for+genome+editing.">CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.</a> Nucleic Acids Research. 42, 401-407 (2014).</li><li>Moravec, C. E., Pelegri, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+accessible+protocol+for+the+generation+of+CRISPR-Cas9+knockouts+using+INDELs+in+zebrafish.">An accessible protocol for the generation of CRISPR-Cas9 knockouts using INDELs in zebrafish.</a> Methods in Molecular Biology. 1920, 377-392 (2019).</li><li>White, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+mRNA+expression+time+course+of+embryonic+development+in+zebrafish.">A high-resolution mRNA expression time course of embryonic development in zebrafish.</a> eLife. 6, 30860 (2017).</li><li>Aanes, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mRNA+sequencing+deciphers+novelties+in+transcriptome+dynamics+during+maternal+to+zygotic+transition.">Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition.</a> Genome Research. 21 (8), 1328-1338 (2011).</li><li>Aken, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ensembl+gene+annotation+system.">The Ensembl gene annotation system.</a> Database: The Journal of Biological Databases and Curation. 2016, (2016).</li><li>Sorlien, E. L., Witucki, M. A., Ogas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+production+and+identification+of+CRISPR/Cas9-generated+gene+knockouts+in+the+model+system+Danio+rerio.">Efficient production and identification of CRISPR/Cas9-generated gene knockouts in the model system Danio rerio.</a> Journal of Visualized Experiments: JoVE. (138), e56969 (2018).</li><li>Kotani, H., Taimatsu, K., Ohga, R., Ota, S., Kawahara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=efficient+multiple+genome+modifications+induced+by+the+crRNAs,+tracrRNA+and+Cas9+protein+complex+in+zebrafish.">efficient multiple genome modifications induced by the crRNAs, tracrRNA and Cas9 protein complex in zebrafish.</a> PloS One. 10 (5), 0128319 (2015).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> Journal of Visualized Experiments: JoVE. (25), e1115 (2009).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+into+zebrafish+embryos.">Microinjection into zebrafish embryos.</a> Methods in Molecular Biology. 127, 125-132 (1999).</li><li>Biology Mouse, ., Zebrafish, , Chick, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology:+Mouse,+Zebrafish,+and+Chick.+Zebrafish+Microinjection+Techniques.">Biology: Mouse, Zebrafish, and Chick. Zebrafish Microinjection Techniques.</a> JoVE Science Education Database. , (2021).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 203 (3), 253-310 (1995).</li><li>D'Agostino, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+cheap+methodology+for+CRISPR/Cas9+zebrafish+mutant+screening.">A rapid and cheap methodology for CRISPR/Cas9 zebrafish mutant screening.</a> Molecular Biotechnology. 58 (1), 73-78 (2016).</li><li>Baars, D. L., Takle, K. A., Heier, J., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ploidy+manipulation+of+zebrafish+embryos+with+Heat+Shock+2+treatment.">Ploidy manipulation of zebrafish embryos with Heat Shock 2 treatment.</a> Journal of Visualized Experiments: JoVE. (118), e54492 (2016).</li><li>Nair, S., Lindeman, R. E., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oocyte+culture-based+manipulation+of+zebrafish+maternal+genes.">In vitro oocyte culture-based manipulation of zebrafish maternal genes.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 242 (1), 44-52 (2013).</li><li>Kushawah, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas13d+induces+efficient+mRNA+knockdown+in+animal+embryos.">CRISPR-Cas13d induces efficient mRNA knockdown in animal embryos.</a> Developmental Cell. 54 (6), 805-817 (2020).</li><li>Kroeger, P. T., Poureetezadi, S. J., McKee, R., Jou, J., Miceli, R., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+haploid+zebrafish+embryos+by+in+vitro+fertilization.">Production of haploid zebrafish embryos by in vitro fertilization.</a> Journal of Visualized Experiments: JoVE. (89), e51708 (2014).</li><li>Xiao, T., Roeser, T., Staub, W., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GFP-based+genetic+screen+reveals+mutations+that+disrupt+the+architecture+of+the+zebrafish+retinotectal+projection.">A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection.</a> Development. 132 (13), 2955-2967 (2005).</li><li>Riemer, S., Bontems, F., Krishnakumar, P., G&#246;mann, J., Dosch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+Bucky+ball-GFP+transgene+visualizes+germ+plasm+in+living+zebrafish.">A functional Bucky ball-GFP transgene visualizes germ plasm in living zebrafish.</a> Gene Expression Patterns: GEP. 18 (1-2), 44-52 (2015).</li><li>Moreno-Mateos, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPRscan:+designing+highly+efficient+sgRNAs+for+CRISPR-Cas9+targeting+in+vivo.">CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.</a> Nature Methods. 12 (10), 982-988 (2015).</li></ol>

The protocol presented in this manuscript allows for the identification and primary molecular characterization of a maternal-effect phenotype in a single generation instead of the multiple generations required for both forward and reverse genetic techniques. Currently, the role of many maternally expressed genes is unknown. This lack of knowledge is partly due to the extra generation required to visualize a phenotype when identifying maternal-effect genes. In the past, the rapid identification of maternal-effect genes in...

A pool of maternally deposited products (e.g., RNAs, proteins, and other biomolecules) is necessary for all early cellular processes until the embryo&#39;s zygotic genome is activated1. The premature depletion of these products from the oocyte is typically embryonic lethal. Despite the importance of these genes in development, the role of many maternally-expressed genes is currently unknown. Advancement in gene-editing technology in zebrafish, such as CRISPR-Cas9, enables the targeting of maternally-expressed genes2,3,4. However, the identification of a maternal-effect phenotype requires an extra generation when compared to a zygotic phenotype, thus requiring more resources. Recently, the biallelic editing capability of CRISPR-Cas9 has been used to screen for embryonic phenotypes in somatic tissues of injected (F0) embryos, known as &#34;crispants&#34;5,6,7,8,9,10. The crispant technique permits resource-efficient screening of candidate genes in somatic cells, facilitating understanding of specific aspects in development. The protocol described in this paper allows for the identification of maternal-effect phenotypes, or &#34;maternal crispants,&#34; in a single generation11. This scheme is attainable by multiplexing guide RNAs to a single gene and promoting biallelic editing events in the germline. These maternal crispant embryos can be identified by gross morphological phenotypes and undergo primary characterization, such as labeling for cell boundaries and DNA patterning11. Combined analysis of the observable phenotype and basic molecular characterization of the induced INDELs allows for the prediction of the targeted gene&#39;s role in early development.
In zebrafish, during the first 24 h post-fertilization (hpf), a small group of cells develops into the primordial germ cells, a precursor to the germline12,13,14,15. In clutches laid by F0 females, the proportion of maternal crispant embryos recovered depends on how many germ cells contain a biallelic editing event in the targeted gene. In general, the earlier the editing event occurs in the embryo, the higher the probability of CRISPR-Cas9 mutations being observed in the germline. In most cases, the phenotypes of maternal crispant embryos come from the loss of function in the two maternal alleles present in the developing oocyte. As the oocyte finishes meiosis, one of the maternal alleles is extruded from the embryo via the polar body, while the other allele becomes incorporated into the maternal pronucleus. The sequencing of multiple maternal crispant haploids will represent a mixture of the mutations (insertions and/or deletions (INDELs)) present in the germline that contribute to the phenotype11.
The following protocol describes the necessary steps to create CRISPR-Cas9 mutations in maternal-effect genes and identify the corresponding phenotype using a maternal crispant approach (Figure 1). Section one will explain how to effectively design and create guide RNAs, while sections two and three contain critical steps for creating maternal crispants by microinjection. After injecting the CRISPR-Cas9 mixture, injected embryos are screened for somatic edits via PCR (section four). Once the injected F0 embryos develop and reach sexual maturity, the F0 females are crossed to wild-type males, and their offspring are screened for maternal-effect phenotypes (section five). Section six includes instructions on making maternal crispant haploids that can be combined with Sanger sequencing to identify the CRISPR-Cas9-induced INDELs. In addition, the Discussion contains modifications that can be made to the protocol to increase the sensitivity and power of this method.

determining the role of maternally-expressed genes in early development with maternal crispants

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary for development until zygotic genome activation. Typically, genetic targeting of these maternal factors requires an additional generation to identify maternal-effect phenotypes, hindering the ability to determine the role of maternally-expressed genes during development. The discovery of the biallelic editing capabilities of CRISPR-Cas9 has allowed screening of embryonic phenotypes in somatic tissues of injected embryos or "crispants," augmenting the understanding of the role zygotically-expressed genes play in developmental programs. This article describes a protocol that is an extension of the crispant method. In this method, the biallelic editing of germ cells allows for the isolation of a maternal-effect phenotype in a single generation, or "maternal crispants." Multiplexing guide RNAs to a single target promotes the efficient production of maternal crispants, while sequence analysis of maternal crispant haploids provides a simple method to corroborate genetic lesions that produce a maternal-effect phenotype. The use of maternal crispants supports the rapid identification of essential maternally-expressed genes, thus facilitating the understanding of early development.

The experimental approach described in this protocol allows for the identification of maternal effect phenotypes in a rapid, resource-efficient manner (Figure 1).
Generating maternal crispants: 
When designing the four guide RNAs to target a single candidate maternal-effect gene, special consideration should be given to where the guide RNAs will bind to DNA. In general, they should all be clustered together with minimal to no overlapping regi...

Watch this Scientific Journal Video about Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants at JoVE.com

Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants

Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary ...

The role of many maternally-expressed genes during early development is currently unknown. This maternal CRISPR technique allows for the rapid identification of maternal effect genes and their role in development. Multiplexing guide RNAs to a single gene, allows researchers to identify novel maternal effect phenotypes in a single generation.This research is beneficial to understand the function of mRNA transcripts in the gametes, which are necessary for early embryogenesis. The key to this technique is to micro inject the embryos early in the one cell stage, and check to see of the majority of the guide RNAs can make somatic mutations in the injected embryos. To create a guide RNA template for each gene specific oligonucleotide, analyte to the constant oligonucleotide and fill in the overhangs with T4 DNA polymerase.After the four guide RNA templates are assembled, purify and concentrate them together using a DNA cleanup and concentrator kit. Synthesize the sgRNA mixture from the pooled guy RNA template, using an in-vitro T7 transcription kit, and perform the in-vitro transcription as described in the manuscript. To verify the integrity of the sgRNA, cast a 1%agarose 0.5 micrograms per milliliter, ethidium bromide Tris-Borate-EDTA gel.Place the solidified gel in Tris-Borate-EDTA running buffer. Mix one microliter sgRNA mixture, and one microliter RNA gel loading buffer. Load this sample in the gel and run the gel at 100 volts for five minutes.Visualize the bands using ultraviolet light. Set up wild type crosses and zebrafish mating boxes. Keep the male and female fish in the same tank, but separate them using a mating box divider, or as shown here, by placing the female inside an egg laying insert.After the injection cocktail is assembled, allow the male and female to mate by removing the mating box divider, or as shown here, by placing the male in the same egg laying insert as the female. To synchronize the embryos, collect them after 10 minutes using a plastic strainer, and rinse them into a Petri dish using 1XE3 media. Remove 10 to 15 embryos and place them into a separate Petri dish to be kept as un-injected controls.Transfer the remaining embryos into the injection plate wells. Inject one nanoliter protein sgRNA solution, into the developing blasto disc of a one cell embryo. Use forceps to break the tip of the clogged needle and recalibrate the needle to eject a one nanoliter bolus.The next day after injection, collect six healthy injected embryos and two control embryos from the un-injected plate. Place each embryo individually into a single well of a PCR strip tube and label the top of the tubes. Design unique screening primers for each guide site to amplify a 100 to 110 base paired DNA fragment that includes the CRISPR-Cas9 target site.If possible, place the target site in the middle of the amplified fragment, allowing for the identification of larger deletions. For each of the four guide target sites, set up eight 25 microliters PCR reactions using five microliters of the prepared single embryogenomic DNA and 20 microliters of the PCR mix, and guide specific screening primers mixture, to identify somatic mutations in the target site. Cast a 2.5%agarose, 0.5 micrograms per milliliter ethidium bromide Tris-Borate-EDTA gel using combs that create approximately 0.625 centimeter wide wells.Then place the solidified gel into the electrophoresis chamber containing Tris-Borate-EDTA running buffer. Add five microliters of 6X loading dye to the PCR product. Load 25 microliters of this mixture into the gel, ensuring that the injected and control samples are running on the same gel row.After all the samples are loaded, add five microliters of ethidium bromide per one liter of Tris-Borate-EDTA running buffer, to the positive end of the gel box. Run the gel at 120 volts until the DNA bans resolve or the DNA has approached the end of the lane. To identify the maternal effect phenotypes and maternal CRISPR's embryos, set up the F0 injected females against wild type males and control wild type crosses in standard zebra fish mating boxes during the afternoon before the experiment.Place the male and female fish in the same tank, but separate them with a mating box divider, or place the female inside an egg laying insert. On the morning of the experiment, allow the male and female to start mating by removing the mating box divider, or placing the male in the same egg laying insert as the female. Collect the embryos every 10 minutes by moving the egg laying insert into a new mating tank bottom that contains fresh system water, and label the tank with a tag identifying the individual F0 female.Take the old mating tank and pour the water through a T-strainer to collect the embryos from one individual 10 minutes clutch. Under a dissecting microscope with a transcendent light source, observe the embryos undergoing development every hour, for the first six to eight hours, and daily for the next five days. Move the potential maternal CRISPR embryos to a Petri dish that contains 1XE3 media, and assay for morphological phenotype at 24 hours post fertilization, and viability, at five days post fertilization.The afternoon before the experiment, set up mating pairs of F0 females by keeping the wild type males physically separated from the females, using a mating box divider or placing the female in the egg laying insert. On the morning of the experiment, remove the physical partition to initiate the mating. At the first sign of egg laying, interrupt breeding by separating the male and F0 females, and keep each separated F0 female in individual mating boxes.After in-vitro fertilization, allow the haploid embryos to develop until the maternal CRISPR phenotype is observed, and place those embryos into a different Petri dish. Once the maternal CRISPR haploid embryos have been identified, allow them to develop for at least six hours post fertilization. To extract the genomic DNA from at least 10 maternal CRISPR haploid embryos, place a single haploid embryo into an individual well of a PCR strip tube, remove excess E3 media from the well, and add 50 microliters of 50 millimolar sodium hydroxide.Incubate the embryos for 20 minutes at 95 degrees Celsius, and cool them down to four degree Celsius. Then add five microliters of one molar Tris-hydrochloric acid with pH 7.5, and vortex for five to 10 seconds. To identify which guide sites contain indels, design sequencing primers to amplify a DNA fragment including all four CRISPR/Cas9 target sites.Set up two 25 microliters of PCR reactions per embryo, using five microliters of the prepared genomic DNA, and the sequencing primers. After the PCR is finished, purify and concentrate the two samples using a DNA cleanup and concentrator kit, and submit the DNA fragment to Sanger sequencing, using the forward and reverse sequencing primers. Guide RNAs should all be clustered together with minimal to no overlapping regions between guide RNAs during maternal CRISPR's generation.If indels were created, a smear should be observed in the injected samples on an agarose gel, but not in the un-injected control. The maternal CRISPR method can be used to phenocopy known maternal effect mutations, such as motley, tmi, and aura. To identify the genetic lesions that contribute to the maternal CRISPR-phenotype, UV treated sperm and in-vitro fertilization are combined to create maternal CRISPR haploids.The creation of a haploid allows Sanger sequencing of the maternal allele, and identification of maternal CRISPR indels. When identifying an unmaternal effect phenotype, is essential that you are able to observe it in multiple F0 females, because it increases the odds that the phenotype is caused by loss of function, not off target or non-specific effects. After a maternal effect phenotype has been identified, the maternal CRISPR embryos can be used to examine the effects on various aspects of early embryogenesis, such as the sinus skeleton or DNA segregation.This allows researchers to understand the molecular cause of the phenotype. This method directly tests the function of unknown maternally expressed genes in a single generation, expediting our understanding of processes, acting during early embryogenesis.

Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...