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)

<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. 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The authors declare no competing financial interests.

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 zebrafish could be achieved by injecting translation-blocking morpholino oligonucleotides into cultured oocytes32. This method was proven successful by phenocopying multiple known maternal-effect genes, but manipulating an immature oocyte can be a delicate, time-consuming experiment. Maternal RNAs can also be targeted for degradation using CRISPR-RfxCas13d complexes, but the injection of these complexes into the one-cell embryo cannot target maternally provided protein33. More recently, it has been discovered that CRISPR-Cas9 can induce biallelic mutations in the germline, allowing for the rapid identification of novel maternal-effect genes in a single generation11.
This protocol includes several critical steps that contribute to the recovery of maternal crispant embryos. In theory, because germ cells are specified in early embryonic development, the earlier a DNA lesion is created in a target gene, the higher the probability that a cell containing mutations will become part of the germline. This method uses Cas9 protein injected into the developing blastodisc of a one-cell embryo to increase the probability of edits in the germline. Another critical factor that affects the percentage of recovered maternal crispant embryos is the efficiency of the guide RNAs in creating genetic edits in target sites. This procedure includes a section on determining the ability of guide RNAs to create somatic INDELs at 24 hpf by PCR. If a guide RNA fails to make somatic edits, it has a low probability of producing edits in the germline at a high enough rate to generate a maternal crispant. This protocol directs the user to test somatic edits, which should be visible in three or more guide sites.
After observing the phenotype of maternal crispant embryos, the genetic lesion(s) contributing to the phenotype can be analyzed at the sequence level via IVF with UV-treated sperm. To acquire enough starting material for PCR, maternal crispant haploid embryos should develop for at least six to eight hours, allowing multiple cycles of DNA replication to occur. The genomic DNA should also be extracted in 50 &#181;L of 50mM NaOH to concentrate the DNA. If the maternal crispant haploid embryos cannot survive for 6-8 h, collect the embryos at an earlier time point. To account for the embryo undergoing fewer cycles of DNA replication, extract the DNA in a smaller volume of 50 mM NaOH while maintaining the same proportion of NaOH and Tris-HCL. Another option is to concentrate the extracted DNA by using a DNA clean-up and concentrator kit. After the DNA has been extracted, the PCR fragment used for sequencing should include all four target sites, if possible. This fragment will allow for the identification of large deletions that span multiple guide sites in maternal crispant embryos.
When collecting maternal crispant haploids for Sanger sequencing, collect all the haploids that show the phenotype and send a minimum of 10 unique haploid embryo samples to Sanger sequencing. The sequencing of multiple haploid embryos per clutch will allow for the identification of multiple INDELs found in the germline. Past sequencing data of maternal crispant haploids have shown that multiple alleles can be identified in a set of maternal crispant haploids11. However, these alleles are not recovered in the expected 1 to 1 ratio11. The sequencing of multiple embryos will also help support the idea that the phenotype is caused by a CRISPR-Cas9 INDEL in the target gene. Any wild-type sequence observed in sequenced haploid embryos that show a specific phenotype will suggest that the phenotype is not associated with the targeted gene. Any novel maternal-effect phenotypes identified through targeting previously uncharacterized genes should also be confirmed by establishing a stable line using the sibling F0 males11.
In some cases, it may be challenging to identify INDELs via haploid analysis where the identified maternal crispant phenotype has a certain phenotypic characteristic. For example, maternal crispant haploid embryos that appear to be unfertilized or lysis during the cleavage stage may be impossible to select. Maternal crispant embryos with axis extension defects similar to those corresponding to the haploid syndrome may also not be distinguishable for analysis when generating maternal haploids34. In such cases, the researcher is advised to conduct the analysis directly using stable lines for gene-phenotype confirmation.
One limitation of the current maternal crispant protocol is that it only identifies maternal-effect genes by gross morphological defects. To increase the method&#39;s sensitivity and make it more specific for certain aspects of early development, transgenic reporters could be used to highlight specific structures or cell types, as has been done for other screens9,10,35. For example, the CRISPR-Cas9 mixture could be injected into the Buc-GFP transgenic line to identify non-lethal genes that regulate the formation and development of primordial germ cells36. Another limitation of the maternal crispant protocol is that, though useful for genes solely expressed during early development, it may not be effective for genes with both maternal and zygotic function since the CRISPR-Cas9 targeting of the gene could be lethal to the developing embryo. To study the maternal function of genes expressed throughout development, Cas9 activity could be targeted to the germline37, thus, leaving the somatic function of the gene unaffected and permitting the survival of the F0 females to adulthood.
Maternal crispants are an effective tool to identify novel maternal-effect genes necessary for early development. The combination of multiplexing guide RNAs to a single target and the biallelic editing ability of Cas9 allows the maternal-effect phenotype to be observed in a single generation, avoiding multi-generation breeding schemes typically needed when performing targeted gene editing. The bypassing of multiple generations decreases the amount of space and resources necessary to identify maternal-effect genes.
In addition to identifying new maternal-effect genes, this protocol permits any zebrafish laboratory to phenocopy and study any known maternal-effect mutant without establishing and maintaining stable lines, facilitating detailed analysis of genetic pathways. In principle, this maternal crispant approach can also determine the function of maternal products in non-genetic model species.

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 regions between guide RNAs at the start of the first predicted protein domain (Figure 2A). Targeting the guide RNAs to this domain increases the chance that both in-frame and out-of-frame INDELs will affect the protein&#39;s function. Other variables that should be considered when designing guide RNAs are cutting efficiency and the number of off-target sites in the genome.
After injecting the CRISPR-Cas9 solution, the somatic activity of Cas9 can be determined by running a small PCR fragment, approximately 100 bp, on an agarose gel. If INDELs were created in the injected embryo, a smear should be observed in the injected samples but not the uninjected control (Figure 2B). Each guide site should be tested independently for Cas9 activity in somatic cells. If smears are seen in at least three guide sites, the sibling injected embryos should be grown up and screened for maternal crispant phenotypes.
Identification of maternal crispants: 
To determine if maternal crispants are created in natural crosses, the embryos from an F0 female fish can be compared to time-matched wild-type controls to observe any changes in early development. F0 clutches should also be scored at 24 hpf and 5 days post-fertilization to examine the development of the basic body plan and viability, respectively, to identify maternal factors that could regulate later stages of embryonic development. Identifying a shared phenotype in clutches from different F0 females facilitates distinguishing effects caused by the loss of function of a target gene from off-target or non-specific effects.
Additionally, clutches containing maternal crispants are typically mosaic (i.e., they include both phenotypical wild-type and maternal crispant embryos), allowing wild-type embryos to act as an internal control for variables such as fertilization timing and developmental rate. On average, clutches from F0 females will contain approximately 69% maternal crispant embryos, with clutches containing up to 100% maternal crispant embryos observed11.
After identifying maternal crispant embryos, they can be used for primary molecular characterization, i.e., immunolabeling for cell boundaries or staining of DNA with DAPI, which can provide insight into the cellular nature of the affected developmental process11. The maternal crispant method can also be used to phenocopy known maternal-effect mutations, such as motley, tmi, and aura (Figure 3)11.
Sequencing of maternal crispant haploids: 
To identify the genetic lesion(s) that contribute to the maternal crispant phenotype, UV-treated sperm and IVF are combined to create maternal crispant haploids (Figure 4). UV-treated sperm provides a centriole but does not contribute paternal DNA, thus permitting cellular division to occur with only maternal genomic material. The creation of a haploid allows Sanger sequencing of the maternal allele and identification of maternal crispant INDELs (Figure 4). On average, two alleles per clutch of maternal crispant haploid were observed. The INDELs identified via Sanger sequencing include edits both in single guide sites and deletions spanning multiple guide sites (Figure 4C, Table 3). A survey of maternal crispant haploid INDELs from different F0 females shows that the same guide sites are edited in multiple embryos, and most of the recovered mutations are premature stop codons (Table 3)11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63177/63177fig01.jpg" /> 
Figure 1: Maternal crispant workflow. To create a maternal crispant, begin by designing four gRNAs that target the first active domain of the gene. 1) Then synthesize the four gRNAs in a single reaction. 2) After synthesizing and purifying the gRNAs, create a CRISPR-Cas9 cocktail and inject it into the blastodisc of a single-cell embryo. 3) Next, screen the injected embryos for somatic mutations using PCR and gel electrophoresis. If INDELs were created in injected samples, a smear would appear in the injected embryos, in contrast to the tight band of the wild-type control. 4) Allow the siblings of the injected embryos to grow for 3-6 months to reach sexual maturity. After sexual maturity is reached, cross an F0 injected female against a wild-type male. The resulting progeny can be a mixture of wild-type and maternal crispant embryos. Identify an F0 injected female whose embryos display the maternal-effect phenotype. 5) To identify the lesions that contribute to the phenotype, IVF is performed using UV-treated sperm to create maternal crispant haploids for Sanger sequencing. <a href="https://www.jove.com/files/ftp_upload/63177/63177fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63177/63177fig02.jpg" /> 
Figure 2: Generation of INDELs in targeted genes. (A) Gene structure diagram showing hypothetical protein domains (light and dark purple blocks), location of gRNAs (red lines), and PAM sites (red stars). The gRNAs are targeted to the first active domain. Exons are shown as blocks, and introns are shown as lines. (B) Smears in a 2.5% agarose gel are indicative of INDELs in somatic cells in injected embryos. <a href="https://www.jove.com/files/ftp_upload/63177/63177fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63177/63177fig03.jpg" /> 
Figure 3: Maternal crispants recapitulate the phenotype of known maternal-effect mutations. Representative comparison of live, time-matched wild-type (left column), known maternal mutants (middle column), and maternal crispant embryos (right column). (A) motley/birc5b, (B) tmi/prc1l, (C) aura/mid1ipIl mutants/maternal crispants show defects in cytokinesis in early embryonic divisions, leading to fully syncytial blastula (A, B), or partially acellular embryos (C, white box). <a href="https://www.jove.com/files/ftp_upload/63177/63177fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63177/63177fig04.jpg" /> 
Figure 4: Using maternal crispant haploids to sequence CRISPR-Cas9-induced mutations. (A) An F0 injected female crossed against a wild-type male results in a diploid embryo with a maternal effect phenotype. (B) IVF is performed using UV-treated sperm to create maternal crispant haploids, allowing for the sequencing and analysis of induced INDELs in the maternal allele. (C) Representative sequencing of birc5b maternal crispant haploid showing a large deletion between guide sites 3 and 4 (boxed). <a href="https://www.jove.com/files/ftp_upload/63177/63177fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">PCR Mix</td>
		</tr>
		<tr>
			<td colspan="3">Add</td>
		</tr>
		<tr>
			<td></td>
			<td>sterile H2O</td>
			<td>171.12 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>MgCl2 (1 M)</td>
			<td>0.393 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>Tris-HCl (1 M, pH 8.4)</td>
			<td>2.618 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>KCl (1 M)</td>
			<td>13.092 mL</td>
		</tr>
		<tr>
			<td colspan="3">Autoclave for 20 min, then chill the solution on ice. Next add</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160;BSA (100 mg/mL)</td>
			<td>3.468 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>dATP (100 mM)</td>
			<td>0.262 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>dCTP (100 mM)</td>
			<td>&#160;0.262 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>dGTP (100 mM)</td>
			<td>0.262 mL</td>
		</tr>
		<tr>
			<td></td>
			<td>dTTP (100 mM)</td>
			<td>0.262 mL</td>
		</tr>
		<tr>
			<td colspan="3">Aliquot into sterile microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>PCR Recipe</td>
			<td></td>
			<td>per&#160; sample</td>
		</tr>
		<tr>
			<td></td>
			<td>PCR Mix</td>
			<td>17.9 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td>F + R Primer (10 &#181;M)</td>
			<td>0.2 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td>ROH2O</td>
			<td>1.8 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td>Taq DNA Polymerase</td>
			<td>0.1 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td>DNA</td>
			<td>5 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: PCR mix.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Hank&#39;s Solution</td>
		</tr>
		<tr>
			<td>Hank&#39;s Premix</td>
			<td colspan="3">Combine the following in order: (1) 10.0 mL of HS #1, (2) 1.0 mL of HS#2, (3) 1.0 mL of HS#4, (4) 86 mL of ddH2O, (5) 1.0 mL of HS#5. Store all HS Solutions at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Stock Solution #1</td>
			<td colspan="3">8.0 g of NaCl, 0.4 g of KCl in 100 mL of ddH2O</td>
		</tr>
		<tr>
			<td>Hank&#39;s Stock Solution #2</td>
			<td colspan="3">0.358 g of Na2HPO4 anhydrous; 0.60 g of K2H2PO4 in 100 mL of ddH2O</td>
		</tr>
		<tr>
			<td>Hank&#39;s Stock Solution #4</td>
			<td colspan="3">0.72 g of CaCl2 in 50 mL of ddH2O</td>
		</tr>
		<tr>
			<td>Hank&#39;s Stock Solution #5</td>
			<td colspan="3">1.23 g of MgSO4&#183;7H2O in 50 mL of ddH20</td>
		</tr>
		<tr>
			<td>Hank&#39;s Stock Solution #6</td>
			<td colspan="3">0.35 g of NaHCO3 in 10.0 mL of ddH20; make fresh on the day of use</td>
		</tr>
		<tr>
			<td>Hank&#39;s Final Working Solution</td>
			<td colspan="3">Combine 9.9 mL of Hank&#39;s Premix with 0.1 mL of HS Stock #6</td>
		</tr>
	</tbody>
</table>
Table 2: Hank&#39;s solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2"></td>
			<td>birc5b #1</td>
			<td>birc5b #2</td>
			<td>birc5b #3</td>
			<td>prc1l #1</td>
			<td>prc1l #2</td>
		</tr>
		<tr>
			<td colspan="2">Total number of embryos sequenced</td>
			<td>3</td>
			<td>9</td>
			<td>12</td>
			<td>8</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="2">Total number of embryos with INDELs</td>
			<td>3</td>
			<td>9</td>
			<td>12</td>
			<td>8</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="2">Mutation in one site</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="2">Mutations in multiple sites</td>
			<td>3</td>
			<td>9</td>
			<td>12</td>
			<td>8</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="2">Location of INDELs</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Guide site 1</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="2">Guide site 2</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>8</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="2">Guide site 3</td>
			<td>3</td>
			<td>9</td>
			<td>12</td>
			<td>8</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="2">Guide site 4</td>
			<td>3</td>
			<td>9</td>
			<td>12</td>
			<td>0</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="2">Types of INDELs</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">In-frame mutation</td>
			<td>1</td>
			<td>2</td>
			<td>2</td>
			<td>7</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="2">Frame shift mutation</td>
			<td>4</td>
			<td>7</td>
			<td>10</td>
			<td>9</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 3: The location and type of INDELs found in two different sets of maternal crispant haploids: birc5b and prc1l.

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 ...

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2.1K Views.  University of Wisconsin-Madison. 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.

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

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 is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

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

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

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, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

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

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 mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

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 example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

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 increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test 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 expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

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 tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

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 polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

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 ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

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 viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

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

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

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 ...

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

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Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

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