The authors would like to thank Nolan Davis for assistance with videography and video editing.

All experimental procedures were approved by the University of Missouri&#39;s Institutional Animal Care and Use Committee (ACUC protocol #25580) and were performed according to the guidelines set forth in the Guide for the Use and Care of Laboratory Animals.
1. AAV-mediated DNA repair template delivery
<ol>
	<li>Design the DNA construct to contain the desired knock-in sequence in between two homology arms. Typical homology arm lengths are 300-500 bp in length. Ensure that th...

<ol>
	<li>Lau, C. H., Tin, C., Suh, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-based+strategies+for+targeted+transgene+knock-in+and+gene+correction.">CRISPR-based strategies for targeted transgene knock-in and gene correction.</a> Fac Rev. 9, 20 (2020).</li><li>Gutschner, T., Haemmerle, M., Genovese, G., Draetta, G. F., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+Regulation+of+Cas9+during+G1+Enhances+Homology-Directed+Repair.">Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.</a> Cell Rep. 14 (6), 1555-1566 (2016).</li><li>Zhang, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+precise+knockin+with+a+double+cut+HDR+donor+after+CRISPR/Cas9-mediated+double-stranded+DNA+cleavage.">Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.</a> Genome Biol. 18 (1), 35 (2017).</li><li>Miura, H., Quadros, R. M., Gurumurthy, C. B., Ohtsuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easi-CRISPR+for+creating+knock-in+and+conditional+knockout+mouse+models+using+long+ssDNA+donors.">Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.</a> Nat Protoc. 13 (1), 195-215 (2018).</li><li>Gu, B., Posfai, E., Gertsenstein, M., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+large-fragment+knock-in+mouse+models+using+2-cell+(2C)-homologous+recombination+(HR)-CRISPR.">Efficient generation of large-fragment knock-in mouse models using 2-cell (2C)-homologous recombination (HR)-CRISPR.</a> Curr Protoc Mouse Biol. 10 (1), e67 (2020).</li><li>Gu, B., Posfai, E., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+targeted+large+insertions+by+microinjection+into+two-cell-stage+mouse+embryos.">Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.</a> Nat Biotechnol. 36 (7), 632-637 (2018).</li><li>Kurihara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+protein+RAD51+enhances+the+CRISPR/Cas9-mediated+knock-in+efficiency+in+brain+neurons.">DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons.</a> Biochem Biophys Res Commun. 524 (3), 621-628 (2020).</li><li>Kaneko, T., Sakuma, T., Yamamoto, T., Mashimo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+knockout+by+electroporation+of+engineered+endonucleases+into+intact+rat+embryos.">Simple knockout by electroporation of engineered endonucleases into intact rat embryos.</a> Sci Rep. 4, 6382 (2014).</li><li>Chen, S., Lee, B., Lee, A. Y., Modzelewski, A. J., He, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+mouse+genome+editing+by+CRISPR+ribonucleoprotein+electroporation+of+zygotes.">Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes.</a> J Biol Chem. 291 (28), 14457-14467 (2016).</li><li>Hashimoto, M., Yamashita, Y., Takemoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+Cas9+protein/sgRNA+into+early+pronuclear+zygotes+generates+non-mosaic+mutants+in+the+mouse.">Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.</a> Dev Biol. 418 (1), 1-9 (2016).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Cas9+protein+into+mouse+zygotes+through+a+series+of+electroporation+dramatically+increases+the+efficiency+of+model+creation.">Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation.</a> J Genet Genomics. 43 (5), 319-327 (2016).</li><li>Troder, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+electroporation+approach+for+efficient+CRISPR/Cas9+genome+editing+in+murine+zygotes.">An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.</a> PLoS One. 13 (5), e0196891 (2018).</li><li>Teixeira, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+mice+zygotes+with+dual+guide+RNA/Cas9+complexes+for+simple+and+efficient+cloning-free+genome+editing.">Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.</a> Sci Rep. 8 (1), 474 (2018).</li><li>Tanihara, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+GGTA1-deficient+pigs+by+electroporation+of+the+CRISPR/Cas9+system+into+in+vitro-fertilized+zygotes.">Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes.</a> BMC Biotechnol. 20 (1), 40 (2020).</li><li>Tanihara, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+CD163-edited+pig+via+electroporation+of+the+CRISPR/Cas9+system+into+porcine+in+vitro-fertilized+zygotes.">Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes.</a> Anim Biotechnol. 32 (2), 147-154 (2021).</li><li>Camargo, L. S. A., Owen, J. R., Van Eenennaam, A. L., Ross, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+one-step+knockout+by+electroporation+of+ribonucleoproteins+into+zona-intact+bovine+embryos.">Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos.</a> Front Genet. 11, 570069 (2020).</li><li>Lin, J. C., Van Eenennaam, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-mediated+genome+editing+of+livestock+zygotes.">Electroporation-mediated genome editing of livestock zygotes.</a> Front Genet. 12, 648482 (2021).</li><li>Betters, E., Charney, R. M., Garcia-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+in+vitro+culture+of+early+rabbit+embryos.">Electroporation and in vitro culture of early rabbit embryos.</a> Data Brief. 21, 316-320 (2018).</li><li>Miyasaka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLICK:+one-step+generation+of+conditional+knockout+mice.">CLICK: one-step generation of conditional knockout mice.</a> BMC Genomics. 19 (1), 318 (2018).</li><li>Epstein, B. E., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+engineered+nucleases+with+adeno-associated+viral+vectors+for+therapeutic+gene+editing.">Combining engineered nucleases with adeno-associated viral vectors for therapeutic gene editing.</a> Adv Exp Med Biol. 1016, 29-42 (2017).</li><li>Gaj, T., Epstein, B. E., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+adeno-associated+virus:+basic+and+clinical+research+applications.">Genome engineering using adeno-associated virus: basic and clinical research applications.</a> Mol Ther. 24 (3), 458-464 (2016).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-READI:+Efficient+generation+of+knockin+mice+by+CRISPR+RNP+electroporation+and+AAV+donor+infection.">CRISPR-READI: Efficient generation of knockin mice by CRISPR RNP electroporation and AAV donor infection.</a> Cell Rep. 27 (13), 3780-3789 (2019).</li><li>Mizuno, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-embryo+gene+cassette+knockin+by+CRISPR/CAS9-mediated+genome+editing+with+adeno-associated+viral+vector.">Intra-embryo gene cassette knockin by CRISPR/CAS9-mediated genome editing with adeno-associated viral vector.</a> iScience. 9, 286-297 (2018).</li><li>Davis, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+DNA+knock-in+using+AAV-mediated+delivery+with+2-cell+embryo+CRISPR-Cas9+electroporation.">Efficient DNA knock-in using AAV-mediated delivery with 2-cell embryo CRISPR-Cas9 electroporation.</a> Front Genome Ed. 5, 1256451 (2023).</li><li>Smirnikhina, S. A., Zaynitdinova, M. I., Sergeeva, V. A., Lavrov, A. 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The CRISPR-Cas genome editing system has revolutionized the field of genetic engineering by allowing the efficient production of both straightforward and complex, customized genetic modifications in a variety of animal species. Frequent refinements and improvements in techniques associated with genome editing further its versatility. Here we have described a new approach using ssAAV-mediated DNA delivery along with 2-cell embryo electroporation of CRISPR-Cas9 reagents into 2-cell rodent embryos. We have demonstrated that...

The development of CRISPR-based genome editing tools has accelerated our ability to efficiently generate new and more sophisticated genetically engineered rat models. Single-guide RNA, along with Cas9 nuclease, is combined to form Ribonucleoprotein (RNP) complexes that target DNA sequences of interest within the genome and result in double-stranded DNA breaks. Because cellular DNA repair mechanisms are error-prone, insertions and deletions (INDELs) are introduced during the repair process that can disrupt a target gene&#39;s function. When there is co-delivery of a desired engineered DNA sequence (repair template) along with genome editing reagents, insertion of the repair template in the region containing the double-stranded DNA break occurs through a process called homology-directed repair (HDR). This is an effective strategy for generating animal models with targeted DNA insertions/substitutions (knock-ins). One limitation is that knock-in sequences are often large in size, which has been shown to reduce gene editing efficiency, thus making generating the desired model more difficult1. Strategies to increase knock-in efficiencies have included linearization of both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) repair templates and chemical modification of DNA repair templates2,3,4. In addition, pronuclear microinjection along with HDR stimulating compounds, application of electrical pulses in conjunction with microinjection, and timed microinjection into 2-cell embryos have all been attempted5,6,7. Despite the success of some of these approaches, the incorporation of DNA sequences larger than 1.0 kb remains technically challenging.
Electroporation, which is a common method for introducing reagents into cultured cell lines, offers an alternative to microinjection for delivering CRISPR-Cas9 components into embryos. Embryo electroporation, first demonstrated in rat embryos8, has since been successfully used as a delivery method in mice9,10,11,12,13, pigs14,15, and other animal model organisms16,17,18. Embryos, suspended in the medium containing CRISPR-Cas9 reagents, are placed into a cuvette or onto a glass slide between two electrodes and subjected to direct pulses of electrical currents. This creates transient openings in the zona pellucida and embryo plasma membrane through which the CRISPR-Cas9 components enter the embryos. Typically, mid-level electrical &#34;poring&#34; pulses are used to create the temporary openings followed by lower level electrical &#34;transfer pulses&#34; that facilitate movement of the negatively charged genome editing components. Embryo electroporation is efficient, has a high throughput, and is easy to perform. However, while embryo electroporation has been shown to be highly successful for the introduction of small (&#60;200 bp) ssDNA repair templates, there are few reports of successful electroporation of larger (&#62;1.0 kb) repair templates13,19. This size restriction represents a major limitation of embryo electroporation for generating knock-in animal models requiring large insertions.
In the context of gene therapy, adeno-associated viruses (AAVs) have long been used as vehicles to deliver genetic material due to their efficient in vivo infectivity of both dividing and non-dividing cells, lack of pathogenicity, and rare genomic integration20,21. Recently, more studies have combined AAVs with CRISPR-Cas9 technology to introduce DNA repair templates and CRISPR reagents22,23,24. This approach allows delivery of larger DNA repair templates without the need for microinjection techniques.
The HDR pathway is more active in the late S and G2 phases of the cell cycle25,26. In studies performed in vitro, significant increases in knock-in efficiency were achieved by delivery of CRISPR-Cas9 RNPs and DNA repair templates into G2-synchronized cells or by restriction of the presence of Cas9 protein to late S and G2 phases using a Cas9-Geminin fusion protein2. Moreover, there is a major zygotic genome activation (ZGA) event which occurs during the extended G2 phase of the 2-cell stage embryo, and this is associated with an open chromatin state. It is speculated that this provides the CRISPR-Cas9 RNPs and repair templates with greater accessibility to the genomic DNA.
Our goal was to build on all these observations, by combining the AAV approach with embryo electroporation to introduce CRISPR-Cas9 RNPs at the 2-cell stage of embryo development. This strategy takes advantage of the larger DNA repair template delivery capacity of AAV, the technical ease of electroporation and the more optimal 2-cell time point for genomic accessibility during embryo development to create an efficient method for targeted genetic engineering of DNA insertions. As highlighted in this protocol, our optimized method allows for the production of targeted knock-in rat models carrying insertions of DNA sequences from 1.2 to 3.0 kb in size without the need for microinjection techniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Leads with alligator clips for electrodes</td>
			<td>NepaGene</td>
			<td>C117</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>MilliporeSigma</td>
			<td>M5310</td>
			<td></td>
		</tr>
		<tr>
			<td>NepaGene21 Super Electroporator&#160;</td>
			<td>NepaGene</td>
			<td>NEPA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum plate electrodes on slide glass</td>
			<td>NepaGene</td>
			<td>CUY501P1-1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>PURedit Cas9 Protein</td>
			<td>MilliporeSigma</td>
			<td>PECAS9</td>
			<td></td>
		</tr>
		<tr>
			<td>sgRNA (chemically-modified)</td>
			<td>Synthego</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ssAAV1 or ssAAV6 packaged DNA repair template</td>
			<td>Vectorbuilder</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

Following the protocol, there is effective AAV-mediated delivery of the DNA repair template allowing for highly efficient HDR after 2-cell embryos are electroporated with CRISPR-Cas9 RNPs. As shown in the video, a successful electroporation process results in bubbles forming on each electrode (Figure 2C) and the impedance remaining within the range of 0.100 and 0.300 k&#8486;. After electroporation it is not uncommon for embryos to swell filling the perivitel...

Watch this Scientific Journal Video about CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation at JoVE.com

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 embryo.

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

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair ...

crispr-cas9-genome-editing-rat-embryos-using-adeno-associated-virus

Research

JoVE Journal

Genetics

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.

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 gene.

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

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

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 ...

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.

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.

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

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

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.

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 editing.

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

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

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

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 ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

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 ...