This research was funded by USDA-NIFA award 2015-67015-23488 to Roger Cone. The authors thank Dr. Ronald Phelps for the description of brood stock selection criteria in the video. Ahmed Elaswad and Karim Khalil would like to thank the Egyptian Cultural and Educational Bureau in Washington DC for funding their Ph.D. research.

This experiment was conducted at the Fish Genetics Research Unit, E. W. Shell Fisheries Research Center, Auburn University, AL. All experimental protocols used in this experiment were approved by the Auburn University Institutional Animal Care and Use Committee (AU-IACUC) before the experiment was initiated. A list of the equipment and supplies used in this experiment can be found in the Materials Table. The following are the steps and procedures for preparation and microinjection of channel catfish one-cell embryos as i...

A detailed protocol for microinjection of channel catfish embryos to achieve gene knockout was presented. Injection of CRISPR/Cas9 protein in the yolk is much simpler, saves time, and does not require extensive training when compared to microinjecting the blastodisc of the one-cell embryo, which is the common technique for most gene transfer and gene editing with fish30,42. The current protocol proved to be successful in inducing indels in channel catfish TICAM 1...

Microinjection is a common laboratory technique used to deliver a small amount of a substance such as DNA, RNA, protein, and other macromolecules into cells or embryos through a glass capillary1. Microinjection is performed using special equipment setup including a microinjector, micromanipulator, and a microscope2. The technique has been used by researchers to genetically modify many organisms through the generation of transgenics, gene knockouts, and gene therapy, with the aim of understanding the dynamics of intracellular components3,4,5.
The channel catfish (Ictalurus punctatus) is the most popular catfish species in aquaculture and recreational fishing activities in the United States. There is an increasing need to study functional genomics in channel catfish, and sequencing of the complete genome of channel catfish enhances the utility of recent advances in genome editing tools6,7. Understanding gene function would not only enrich the research that is being conducted on catfish, but could also lead to more effective genetic improvement programs to enhance the catfish industry. Once critical genes for a given trait of interest are identified, they can be used to genetically improve catfish production through genome editing to generate beneficial alleles, selection for these alleles, suppressing the detrimental alleles, transferring the beneficial alleles through transgenesis, or some combination of these options. Combining the best genes for different commercially beneficial traits from different species in one fish would greatly improve productivity and profitability of fish production operations8.
Gene knockout is a direct method to study gene functions in vivo. Mutations at the DNA level will be inherited by future generations which will facilitate the study of their effects in different generations. Different genome editing tools have been developed including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system9,10,11,12.
The CRISPR/Cas9 system is a powerful, efficient tool that has been used to edit genomic DNA sequences including gene knockout in fish through RNA-guided site-specific DNA cleavage5,13. The system consists of a guide RNA (gRNA), which determines the targeted sequence in the genome and a DNA endonuclease enzyme, Cas9. The CRISPR/Cas9 system can be designed to target any sequence in the genome with several advantages over ZFNs and TALENs: (1) lower cost (2) easier engineering (3) more specific binding of guide RNA to the target sequence, and reduced off-target mutations (4) multiple sequences can be targeted with different gRNA at the same time (5) high mutagenesis rate in genes that could not be mutated by TALENs, and (6) improved germline transmission rate of mutations for up to 6-fold when compared to ZFNs and TALENs14,15,16,17,18.
The main alternative method for microinjection is electroporation, in which electric impulses are applied to embryos or cells to increase membrane permeability and the uptake of biological molecules19,20. Several transgenic fish were generated using electroporation such as medaka (Oryzias latipes), zebrafish (Danio rerio), chinook salmon (Oncorhynchus tshawytscha), channel catfish, sea bream (Sparus sarba), and common carp (Cyprinus carpio)21,22,23,24,25,26.
Electroporation has been used to deliver plasmid DNA, RNA, and Cas9 protein for gene knockout. In mammalian cells, Cas9/gRNA plasmid DNA, Cas9 mRNA/gRNA, and Cas9 protein/gRNA complexes were delivered using electroporation and the mutagenesis rates were highest using Cas9 protein/gRNA complexes for most electroporation conditions tested27. In the ascidian chordate (Ciona intestinalis), TALENs expressing constructs were electroporated into fertilized eggs to induce knockout of multiple genes28. ZFNs expressing plasmid constructs were electroporated to knock out luteinizing hormone in channel catfish29. However, once introduced into the cell, plasmid constructs will need to be transcribed to RNA and translated to functional proteins before they can target the desired DNA sequence, which would likely delay the time of mutagenesis compared to microinjection of RNA/protein. As an embryo is developing, delayed mutagenesis increases the mosaicism of injected founders. In addition, transgenic expression is unlikely to achieve the expression level possible with microinjection, lowering the mutagenesis efficiency.
As a means for introducing genome editing tools into cells, microinjection has several advantages over electroporation. Genome editing molecules can be reliably introduced into cells or embryos through microinjection30. Less injection material is needed. It is easy to determine the amounts of material injected. Higher mutation rates with low mosaicism in the founder fish can be achieved which would improve germline transmission of mutations to F1. Fewer founder fish need to be analyzed to produce the first generation, due to the higher mutation rate. In electroporation, more founder fish need to be analyzed which will increase costs. However, the survival of channel catfish microinjected embryos is lower than electroporated ones, though this can be overcome by injecting more embryos31,32. In channel catfish, survival of yolk-microinjected embryos ranged from 16 to 55%, depending on the genes being targeted and the dosage of gRNA/Cas9 protein32.
Regular microinjection of catfish embryos is technically demanding and time consuming, however, a rapid and efficient CRISPR/Cas9 protein microinjection protocol is presented for channel catfish embryos. This protocol requires less time and expertise since the CRISPR/Cas9 protein solution is injected into the yolk of one cell embryos. Hundreds of fertilized eggs can be injected in 1 h (approximately the time needed for the first cell division to occur). To validate the protocol, two disease susceptibility-related genes were knocked out in channel catfish, the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM1) gene and the rhamnose binding lectin (RBL) gene. TICAM 1 is involved in the signaling pathway initiated by toll-like receptor (TLR) 3. In channel catfish, TICAM 1 was dramatically upregulated following bacterial challenge with Edwardsiella ictaluri, while it was downregulated in blue catfish, a species resistant to E. ictaluri33. RBL plays an important role in early infection with Flavobacterium columnare, the causative agent of columnaris disease, where acute and robust upregulation was recorded in a columnaris-susceptible channel catfish strain compared to a columnaris-resistant strain34.

<table border="0" cellpadding="0" cellspacing="0" width="520">
	<tbody>
		<tr height="100">
			<td height="100" style="height:100px;width:136px;">Reproboost implant</td>
			<td style="width:115px;">Center of Marine Biotechnology</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">Luteinizing hormone releasing hormone analog (LHRHa) for induction of ovulation in channel catfish females</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:136px;">TRICAINE-S</td>
			<td style="width:115px;">Western Chemical. Inc.</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">For sedation of brood stock fish during hormone injection and egg stripping.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:136px;">Phenol red</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:99px;">P0290</td>
			<td style="width:171px;">0.5%, sterile filtered</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Stereo microscope</td>
			<td style="width:115px;">Olympus</td>
			<td style="width:99px;">213709</td>
			<td style="width:171px;">For visualizing the eggs during microinjection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">Microinjector</td>
			<td style="width:115px;">ASI-Applied Scientific Instrumentation&#160;</td>
			<td style="width:99px;">Model MPPI-3</td>
			<td style="width:171px;">For the delivery of the injection material into the embryos</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:136px;">Micromanipulator</td>
			<td style="width:115px;">ASI-Applied Scientific Instrumentation&#160;</td>
			<td style="width:99px;">Model MM33&#160;</td>
			<td style="width:171px;">For holding and controlling the movement of the injection needle.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:136px;">Eppendorf Microloader</td>
			<td style="width:115px;">Eppendorf</td>
			<td style="width:99px;">5242956.003</td>
			<td style="width:171px;">For loading injection solution into microinjection needles.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:136px;">Vertical needle puller</td>
			<td style="width:115px;">David Kopf Instruments</td>
			<td style="width:99px;">Model 720</td>
			<td style="width:171px;">For pulling microinjection needles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:136px;">Cas9 protein</td>
			<td style="width:115px;">PNA Bio Inc.&#160;</td>
			<td style="width:99px;">CP01</td>
			<td style="width:171px;">Recombinant Cas9 protein from Streptococcus pyrogenes.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">Expand High FidelityPLUS PCR System</td>
			<td style="width:115px;">&#160;Roche Diagnostics, USA</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">For PCR and amplification of DNA templates to be used in gRNA preparation</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:136px;">Borosilicate glass capillaries</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">1 mm outer diameter (OD), for making microinjection needles.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Petri dish</td>
			<td style="width:115px;">VWR</td>
			<td style="width:99px;">25384-302</td>
			<td style="width:171px;">For holding the embryos during the microinjection.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">Crisco</td>
			<td style="width:115px;">The J.M. Smucker Company</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">Vegetable shortening for coating spawning pans and petri dishes.&#160;</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:136px;">Holtfreter`s solution</td>
			<td style="width:115px;">Home Made</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">59 mM NaCl, 0.67 mM KCl, 2.4 mM NaHCO3, 0.76 mM CaCl2, 1.67 mM MgSO4 to incubate the microinjected embryos till hatch.&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:136px;">Doxycycline hyclate USP (monohydrate)</td>
			<td style="width:115px;">Letco Medical</td>
			<td style="width:99px;">690904</td>
			<td style="width:171px;">Antibiotic added to Holtfreter&#39;s solution to 10 ppm to prevent bacterial infections.</td>
		</tr>
	</tbody>
</table>

microinjection of crispr/cas9 protein into channel catfish, ictalurus punctatus, embryos for gene editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

To demonstrate the efficiency of the microinjection protocol, gRNAs designed to target the channel catfish toll/interleukin 1 receptor domain-containing adapter molecule (TICAM1) gene and rhamnose binding lectin (RBL) gene were microinjected.
TICAM 1 
DNA sequencing of vectors from individual colonies from pooled, cloned PCR products revealed the indel mutations induced in TICAM 1 gene. The mutation rate wa...

Watch this Scientific Journal Video about Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing at JoVE.com

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

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14.7K ビュー.  Auburn University. CRISPR/Cas9 システムを用いたチャネル ナマズ胚の遺伝子を編集するための簡単かつ効率的なマイクロインジェクション プロトコルが表示されます。このプロトコルでは Rna、ガイドし、Cas9 蛋白質は 1 細胞期胚の卵黄に microinjected いた。このプロトコルは、2 つのチャネル ナマズ免疫関連遺伝子をノックアウトによって検証されています。

ビデオ: Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

microinjection of corn planthopper, peregrinus maidis, embryos for crispr/cas9 genome editing

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline...

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary.  This allowed for the detection of the bacterium using fluorescent...

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

crispr/cas9 ribonucleoprotein-mediated precise gene editing by tube electroporation

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube...

a crispr-cas9 technique for gene editing in t cells

This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell&#39;s non-homologous end-joining mechanism, leading to gene...

A CRISPR-Cas9 Technique for Gene Editing in T Cells

Electroplate the cells using pulse code EH-1-11. Then, incubate 5 million cells per milliliter in R-10, supplemented with five nanograms per milliliter human IL-7 and human Il-15 at 30 degrees Celsius for 48 hours in 12-well plates. After the incubation, proceed with T cell activation and...

generating recombinant avian herpesvirus vectors with crispr/cas9 gene editing

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox...

crispr/cas9 gene editing of hematopoietic stem and progenitor cells for gene therapy applications

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in...

a technique for gene editing in natural killer cells using crispr cas9

This video demonstrates a technique for Cas9 ribonucleoprotein-mediated genetic modification of primary natural killer (NK) cells. A Cas9 ribonucleoprotein, consisting of a Cas9 endonuclease bound to a guide RNA (gRNA) formed by base pairing a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), is introduced into primary natural killer cells via electroporation. The ribonucleoprotein targets and cleaves the host DNA at the target site, leading to gene knockout via modification of the...

A Technique for Gene Editing in Natural Killer Cells Using CRISPR Cas9

While the medium is equilibrating, add 3 to 4 x 106 cells per condition, per 26 microliters of transduction mix into a conical tube, and pellet the cells by centrifugation. Then, wash the cells three times in 12 milliliters of sterile PBS per wash to remove all of the FBS from the cell...

sand fly (phlebotomus papatasi) embryo microinjection for crispr/cas9 mutagenesis

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

crispr-cas9 genome editing of rat embryos using adeno-associated virus (aav) and 2-cell embryo electroporation

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell...

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

development of knock-out muscle cell lines using lentivirus-mediated crispr/cas9 gene editing

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out...

crispr/cas9 gene editing to make conditional mutants of human malaria parasite p. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

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

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...

crispr/cas9 editing of the c. elegans rbm-3.2 gene using the dpy-10 co-crispr screening marker and assembled ribonucleoprotein complexes.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

crispr gene editing tool for microrna cluster network analysis

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single...

qtl mapping and crispr/cas9 editing to identify a drug resistance gene in toxoplasma gondii

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct&#160;in vivo&#160;CRISPR&#160;screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...