Name: silencing the spark: crispr/cas9 genome editing in weakly electric fish
Uploaded: 2019-10-27T14:00:00
Description: Watch this Scientific Journal Video about Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish at JoVE.com

The authors acknowledge the heroic efforts of Monica Lucas, Katherine Shaw, Ryan Taylor, Jared Thompson, Nicole Robichaud, and Hope Healey for help with fish husbandry, data collection, and early protocol development. We would also like to thank the three reviewers for their suggestions to the manuscript. We believe the final product to be of better quality after addressing their comments. This work was funded by support from the National Science Foundation #1644965 and #1455405 to JRG, and the Natural Sciences and Engineering Research Council DG grant to VLS.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University.
1. Selecting sgRNA Targets
NOTE: A protocol is provided for manual design of sgRNAs in step 1.1. This was utilized for scn4aa target selection. An additional protocol is provided to facilitate this process (step 1.2) using the EFISHGENOMICS web portal. It is advised that users select protocol 1.2, which featur...

The phenotypic richness of weakly electric fish, together with a recent proliferation of genomics resources, motivates a strong need for functional genomic tools in the weakly electric fish model. This system is particularly attractive because of the convergent evolution of numerous phenotypic traits in parallel lineages of fish, which are easily kept in the laboratory.
The protocol described here demonstrates the efficacy of the CRISPR/Cas9 technique in lineages of weakly electric fish that e...

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. Two lineages of teleost fish, osteoglossiformes and siluriformes, evolved electroreception in parallel, and five lineages of teleosts (gymnotiformes, mormyrids, and the genera Astroscopus, Malapterurus, and Synodontis) evolved electrogenesis in parallel. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture1,2,3.
This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades, which produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate4, a growing community of scientists has gained tremendous insights into evolution of development1,5,6, systems and circuits neuroscience7,8,9,10, cellular physiology11,12, ecology and energetics13,14,15,16,17, behavior18,19, and macroevolution3,20,21.
More recently, there has been a proliferation of genomic, transcriptomic, and proteomic resources for electric fish1,22,23,24,25,26,27,28. Use of these resources has already produced important insights regarding the connection between genotype and phenotype in these species1,2,3,28,29,30. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools31.
One such tool is the recently developed Clustered Regularly Interspaced Short Palindromic Repeats paired with Cas9 endonuclease (CRISPR/Cas9, CRISPR) technique. CRISPR/Cas9 is a genome editing tool that has entered widespread use in both model32,33,34 and non-model organisms35,36,37 alike. CRISPR/Cas9 technology has progressed to a point where a laboratory capable of basic molecular biology can easily generate gene-specific probes called short guide RNAs (sgRNAs), at a low cost using a non-cloning method38. CRISPR has advantages over other knockout/knockdown strategies, such as morpholinos39,40, transcription activator-like effector nucleases&#160;(TALENs), and zinc finger nucleases (ZFNs), which are costly and time-consuming to generate for every target gene.
The CRISPR/Cas9 system functions to create gene knockouts by targeting a specific region of the genome, directed by the sgRNA sequence, and causing a double-stranded break. The double-stranded break is detected by the cell and triggers endogenous DNA repair mechanisms preferentially using the non-homologous end joining (NHEJ) pathway. This pathway is highly error-prone: during the repair process, the DNA molecule will often incorporate insertions or deletions (indels) at the double-stranded break site. These indels can result in a loss of function due to either (1) shifts in the open reading frame, (2) insertion of a premature stop codon, or (3) shifts in the critical primary structure of the gene product. In this protocol, we utilize CRISPR/Cas9 editing to target point mutations in target genes using the NHEJ in weakly electric fish species. While simpler and more efficient than other techniques, this method of mutagenesis is expected to result in a range of phenotypic severities in F0, which is attributed to genetic mosaicism41,42,43,44.
Selection of Organisms 
For the purposes of facilitating future studies on the comparative genomics of weakly electric fish, a representative species for both gymnotiforms and mormyrids for protocol development needed to be selected. Following discussions during the 2016 Electric Fish meeting in Montevideo, Uruguay, there was community consensus to utilize species that already could be bred in the laboratory and that had genomic resources available. The gymnotiform Brachyhypopomus gauderio and the mormyrid Brienomyrus brachyistius were selected as species that fit these criteria. In both species, natural cues to induce and maintain breeding conditions are easy to mimic in captivity. B. gauderio, a gymnotiform species from South America, has the advantage of low husbandry requirements: fish can be kept at relatively high density in relatively small (4 L) tanks. B. gauderio also has fast generational turnover under captive conditions. Under laboratory conditions, B. gauderio can develop from egg to adult in about 4 months.
B. brachyistius, a species of mormyrid fish from West-Central Africa, breeds readily in captivity. B. brachyistius is readily available through the aquarium trade, has been widely used in many studies, and now has a number of genomic resources available. Their life cycle spans 1&#8722;3 years, depending on laboratory conditions. Husbandry requirements are somewhat more intensive for this species, requiring moderately sized tanks (50&#8722;100 L) due to their aggression during breeding.
Laboratories studying other species of electric fish should be able to easily adapt this protocol as long as the species can be bred, and single cell embryos can be collected and reared into adulthood. Housing, larval husbandry, and in vitro fertilization (IVF) rates will likely change with other species; however, this protocol can be used as a starting point for breeding attempts in other weakly electric fish.
An Ideal Gene Target for Proof of Concept: scn4aa 
Weakly electric mormyrid and gymnotiform fish generate electric fields (electrogenesis) by discharging a specialized organ, called the electric organ. Electric organ discharges (EODs) result from the simultaneous production of action potentials in the electric organ cells called electrocytes. EODs are detected by an array of electroreceptors in the skin to create high-resolution electrical images of the fish&#8217;s surroundings45. Weakly electric fish are also capable of detecting features of their conspecifics&#8217; EOD waveforms18 as well as their discharge rates46, allowing EODs to function additionally as a social communication signal analogous to birdsong or frog vocalizations47.
A main component of action potential generation in the electrocytes of both mormyrid and gymnotiform weakly electric fish is the voltage-gated sodium channel NaV1.42. Non-electric teleosts express two paralogous gene copies, scn4aa and scn4ab, coding for the voltage-gated sodium channel NaV1.430. In both gymnotiform and mormyrid weakly electric fish lineages, scn4aa has evolved rapidly and undergone numerous amino acid substitutions that affect its kinetic properties48. Most importantly, scn4aa has become compartmentalized in both lineages to the electric organ2,3. The relatively restricted expression of scn4aa to the electric organ, as well as its key role in the generation of EODs, makes it an ideal target for CRISPR/Cas9 knockout experiments, as it has minimal deleterious pleiotropic effects. Because weakly electric fish begin discharging their larval electric organs 6&#8722;8 days post fertilization (DPF), targeting of scn4aa is ideally suited for rapid phenotyping following embryo microinjection.

<table>
	<tbody>
		<tr>
			<td>20 mg/mL RNA grade Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
			<td></td>
		</tr>
		<tr>
			<td>50 bp DNA ladder</td>
			<td>NEB</td>
			<td>N3236L</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary with filament</td>
			<td>Sutter Instrument</td>
			<td>BF100-58-10</td>
			<td>(O.D. 1.0mm, I.D. 0.58 mm, 10 cm length)</td>
		</tr>
		<tr>
			<td>Cas9 protein with NLS; 1 mg/mL</td>
			<td>PNA Biology</td>
			<td>CP01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dneasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf FemptoJet 4i Microinjector</td>
			<td>Fisher Scientific</td>
			<td>E5252000021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader Pipette Tips</td>
			<td>Fisher Scientific</td>
			<td>10289651</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Fisher Scientific</td>
			<td>14-824-654</td>
			<td>referred to as &#34;precision glass syringe&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td>referred to as &#34;delicate task wipe&#34; in the protocol</td>
		</tr>
		<tr>
			<td>MEGAscript T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 3</td>
			<td>NEB</td>
			<td>B7003S</td>
			<td>used for in vitro cleavage assay</td>
		</tr>
		<tr>
			<td>OneTaq DNA kit</td>
			<td>NEB</td>
			<td>M0480L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovaprim</td>
			<td>Syndel USA</td>
			<td>https://www.syndel.com/ovaprim-ovammmlu010.html</td>
			<td>referred to as &#34;spawning agent&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>S37440</td>
			<td>referred to as &#34;thermoplastic&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>WPI</td>
			<td>SU-P97</td>
			<td>sutter brand</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable needle- requires customization</td>
			<td>Fisher Scientific</td>
			<td>7803-02</td>
			<td>Customize to 0.7 inches long; point style 4 and angle 25</td>
		</tr>
		<tr>
			<td>T4 DNA polymerase</td>
			<td>NEB</td>
			<td>M0203L</td>
			<td>Use with the 10X NEB buffer that is included</td>
		</tr>
		<tr>
			<td>Teflon coated tools</td>
			<td>bonefolder.com</td>
			<td>T-SPATULA4PIECE</td>
			<td>referred to as &#34;polytetrafluoroethene&#34; in the protocol</td>
		</tr>
	</tbody>
</table>

silencing the spark: crispr/cas9 genome editing in weakly electric fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

The sgRNA target sites were identified within exon 1 of scn4aa in both B. gauderio and B. brachyistius as described in Section 1. The sgRNAs were generated as described in Section 2. Following successful sgRNA selection and synthesis (Figure 1), in vitro cleavage was tested (Figure 2). The sgRNAs demonstrating in vitro cutting were then selected for single cell microinjections.
Adult fish were conditioned fo...

Watch this Scientific Journal Video about Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish at JoVE.com

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

CRISPR/Cas9 gene editing in weakly electric fish, particularly in a comparative context, will enable researchers to test hypotheses about how specific genes and genetic variance contribute to phenotypic variation. CRISPR/Cas9 is an efficient method for generating mutant F-zero embryos. This protocol allows researchers to conduct CRISPR-Cas9 manipulations in two convergently evolved species of weakly electric fish.The key to using this approach with some modifications in other electric fish or other fish species is the availability of transcriptomic or genomic resources to make sgRNAs. Performing the microinjections and visualizing the cells in a 3D location within a 2D plane can be difficult. Obtain a small zebrafish colony for eggs to practice general microinjection techniques.Demonstrating the procedure with Savvas Constantinou will be Jared Thompson, a technician from my laboratory. After housing the fish species of interest under the appropriate breeding conditions, identify female fish that appear gravid. In B.gauderio, the female will have swollen gonads just caudal to the vent.In B.brachyistius females will have swollen bellies and appear deep-bodied. After anesthesia administration, add spawning agent to four times the volume of DPBS in a PCR tube with thorough mixing. The solution will become cloudy.Use a pipette to measure out the calculated dose and dispense the diluted agent unto a piece of thermoplastic before drawing the solution into a precision glass syringe equipped with a 28-gauge 19 to 25-milliliter length beveled needle. Inject the solution into the dorsal trunk muscle at a smooth rate and let the needle sit for two to four seconds before removal. Then, immediately place fish into fresh system water for recovery.For B.gauderio sperm collection, after 24 hours, select a large male fish and as quickly as possible after sedation, dry the fish thoroughly, especially around the head and Vent. Place the dried fish ventral side up, anterior to the left in appropriate holder, covered in an MS222 soaked damp paper towel with the head as parallel with the table as possible. Immediately apply pressure in the caudal to rostral direction with light squeezing immediately over the gonads toward the vent and using a micropipetter with a tip, carefully collect the sperm as it is squeezed from the male in 50-microliter increments.Add aliquot of sperm directly onto 500-microliter volumes of sperm extender solution on ice. Immediately after the collection, place fish into slime coat protection product-treated fresh system water. For B.gauderio egg collection, after drying an a anesthetized B.gauderio female fish, immediately place the fish on its side with the head facing the nondominant hand and apply pressure in the caudal to rostral direction with light squeezing medially over the gonads toward the vent.Using a polytetrafluoroethylene tool, carefully collect the eggs as they are squeezed from the cloaca and quickly place the eggs in a small covered Petri dish. If working alone after squeezing, touch the egg mass to the base of the small Petri dish as they are expelled from the female. Immediately after collecting the eggs, place the fish in slime coat protection product-treated fresh system water.For B.gauderio in vitro fertilization, add 100 microliters of the well mixed sperm SES solution directly over the egg mass, followed by one milliliter of fresh system water. Mix the gametes suspension thoroughly for 30 to 60 seconds before adding an additional one to two milliliters of 0.22-micrometer pore filtered system water to the cells. Label the dish with the in vitro fertilization time and place the dish into a 29-degree Celsius incubator, setting a 50-minute timer to check the progress of the development.During the fertilization period, use a microloader pipette tip to backload two microliters of the injection solution of interest into a microinjection needle and expel the liquid as close to the tip as possible. When a single cell has formed at the animal pole of at least one cell, transfer the dish under a dissecting microscope and use a pair of fine forceps to break the needle tip at a beveled angle toward where the taper of the needle begins to demonstrate rigidity. The bore of the needle should remain as small as possible while maintaining a rigid taper.Identify the developing zygotes under the microscope and use a plastic transfer pipette with a cut tip to place 10 to 20 eggs in as little water as possible onto the edge of the glass microscope slide set in the inverted top of the Petri dish. Using a delicate task wipe, gently press the slide to remove the excess water to allow the eggs to adhere firmly to the edge of the slide. Water will be wicked under the slide and pull eggs against the edge.There should be just enough water to keep the eggs moist while maintaining little standing moisture to avoid egg movement during injection. Align the eggs against the slide vertically, perpendicular to the approaching needle, and use a micromanipulator to position the needle against the chorion. Then, holding the needle at a roughly 45-degree angle, insert the tip first into the chorion and then, into the single cell, injecting through the yolk if possible.Remove any broken eggs during the injection with fine forceps. When all of the eggs has been injected, use a squirt bottle of 0.22-micrometer pore filtered system water to gently flush the eggs from the injection stage into a new 100-millimeter diameter Petri dish. Following a successful short guide RNAs selection and synthesis, in vitro cleavage can be tested for selection for single cell microinjections.After PCR clean up and cloning, in this representative experiment, CRISPR-Cas9 induced mutations were identified in individual B.gauderio and B.brachyistius clones with strong electric organ discharge amplitude reduction, whereas uninjected controls displayed only referenced genotypes. Visualization of the electric organ discharge amplitude between confirmed CRISPR mutants and agent size matched uninjected controls demonstrated that both SCN-4 AA mutant B.brachyistius and B.gauderio embryos had significantly lower electric organ discharge amplitudes than controls. When performing the micro injections, move as quickly and deliberately as possible to maximize the number of single-cell injected embryos that survive.Do not waste time with difficult eggs. Once successful mutants have been identified, traditional breeding methods can create stable mutant lines, which eliminate CRSPR associated mosaicism concerns. This protocol will allow researchers to conduct comparative genomic studies with functional validation in two convergently evolved species of weakly electric fish.

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JoVE Journal

Biology

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...