This research was supported by a Canadian Institutes of Health Research Project grant (T.W.C.) and Natural Sciences and Engineering Research Council of Canada Discovery grants (T.W.C.).

Studies using zebrafish were conducted in agreement with the policies and procedures of the Simon Fraser University Animal Care Committee and the Canadian Council of Animal Care and were completed under protocol # 1264K-18.
1. Design of CRISPR components for precise edits
<ol>
	<li>To design the two-sgRNA guides that will be used to excise the sequence containing the KI target site, first identify the zebrafish ortholog for the gene of interest. 
	NOTE: <strong class="x...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+editing+of+the+Zebrafish+genome+made+simple+and+efficient.">Precise editing of the Zebrafish genome made simple and efficient.</a> Developmental Cell. 36 (6), 654-667 (2016).</li><li>Albadri, S., Del Bene, F., Revenu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+using+CRISPR/Cas9-based+knock-in+approaches+in+zebrafish.">Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish.</a> Methods. 121-122, 77-85 (2017).</li><li>Armstrong, G. A. B., 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=Homology+directed+knockin+of+point+mutations+in+the+zebrafish+tardbp+and+fus+genes+in+ALS+using+the+CRISPR/Cas9+system.">Homology directed knockin of point mutations in the zebrafish tardbp and fus genes in ALS using the CRISPR/Cas9 system.</a> PLoS One. 11 (3), 0150188 (2016).</li><li>Bai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+precise+genome+modification+by+a+long+ssDNA+template+in+zebrafish.">CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish.</a> BMC Genomics. 21 (1), 67 (2020).</li><li>de Vrieze, 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=Efficient+generation+of+knock-in+zebrafish+models+for+inherited+disorders+using+CRISPR-Cas9+ribonucleoprotein+complexes.">Efficient generation of knock-in zebrafish models for inherited disorders using CRISPR-Cas9 ribonucleoprotein complexes.</a> International Journal of Molecular Sciences. 22 (17), 9429 (2021).</li><li>Eschstruth, A., Schneider-Maunoury, S., Giudicelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+zebrafish+knock-in+reporter+lines+in+the+nefma+gene+by+Cas9-mediated+homologous+recombination.">Creation of zebrafish knock-in reporter lines in the nefma gene by Cas9-mediated homologous recombination.</a> Genesis. 58 (1), 23340 (2020).</li><li>Irion, U., Krauss, J., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+and+efficient+genome+editing+in+zebrafish+using+the+CRISPR/Cas9+system.">Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system.</a> Development. 141 (24), 4827-4830 (2014).</li><li>Kimura, Y., Hisano, Y., Kawahara, A., Higashijima, S. <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+knock-in+transgenic+zebrafish+carrying+reporter/driver+genes+by+CRISPR/Cas9-mediated+genome+engineering.">Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.</a> Scientific Reports. 4 (1), 6545 (2014).</li><li>Levic, D. S., Yamaguchi, N., Wang, S., Knaut, H., Bagnat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knock-in+tagging+in+zebrafish+facilitated+by+insertion+into+non-coding+regions.">Knock-in tagging in zebrafish facilitated by insertion into non-coding regions.</a> Development. 148 (19), (2021).</li><li>Prykhozhij, S. V., 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=Optimized+knock-in+of+point+mutations+in+zebrafish+using+CRISPR/Cas9.">Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.</a> Nucleic Acids Research. 46 (17), 102 (2018).</li><li>Wierson, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeted+integration+directed+by+short+homology+in+zebrafish+and+mammalian+cells.">Efficient targeted integration directed by short homology in zebrafish and mammalian cells.</a> eLife. 9, 53968 (2020).</li><li>Boel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+homology-directed+repair+by+ssODNs+in+zebrafish+induces+complex+mutational+patterns+resulting+from+genomic+integration+of+repair-template+fragments.">CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments.</a> Disease Models &amp; Mechanisms. 11 (10), (2018).</li><li>Tessadori, 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=Effective+CRISPR/Cas9-based+nucleotide+editing+in+zebrafish+to+model+human+genetic+cardiovascular+disorders.">Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders.</a> Disease Models &amp; Mechanisms. 11 (10), (2018).</li><li>Zhang, Y., Zhang, Z., Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+platform+for+generating+somatic+point+mutations+with+germline+transmission+in+the+zebrafish+by+CRISPR/Cas9-mediated+gene+editing.">An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing.</a> The Journal of Biological Chemistry. 293 (17), 6611-6622 (2018).</li><li>Vornanen, M., Hassinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+heart+as+a+model+for+human+cardiac+electrophysiology.">Zebrafish heart as a model for human cardiac electrophysiology.</a> Channels. 10 (2), 101-110 (2016).</li><li>Nemtsas, P., Wettwer, E., Christ, T., Weidinger, G., Ravens, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+heart+as+a+model+for+human+heart?+An+electrophysiological+study.">Adult zebrafish heart as a model for human heart? 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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=Optimisation+of+embryonic+and+larval+ECG+measurement+in+zebrafish+for+quantifying+the+effect+of+QT+prolonging+drugs.">Optimisation of embryonic and larval ECG measurement in zebrafish for quantifying the effect of QT prolonging drugs.</a> PLoS One. 8 (4), 60552 (2013).</li><li>Yu, 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=Evolving+cardiac+conduction+phenotypes+in+developing+zebrafish+larvae:+Implications+to+drug+sensitivity.">Evolving cardiac conduction phenotypes in developing zebrafish larvae: Implications to drug sensitivity.</a> Zebrafish. 7 (4), 325-331 (2010).</li><li>Hurst, R. 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The authors have no conflicts of interest to disclose.

The engineering of precise gene edits using CRISPR-Cas9 is challenged by the low efficiencies of HDR mechanisms and their efficient detection. Here, a CRISPR-Cas9-based two-sgRNA exon replacement approach is described that produces precise edits in zebrafish with straightforward visual detection of positive edits. The efficacy of this approach is demonstrated by generating precise edits in the zkcnh6a gene. This paper shows how cardiac function in gene-edited zebrafish larvae may be assessed using non-invasive p...

CRISPR-based gene editing strategies in animal models enable the study of genetically heritable disease, development, and toxicology at the whole-organism level1,2,3. Zebrafish provide a powerful model that is closer in numerous physiological aspects to humans than murine or human-derived cell models4. An extensive array of genetic tools and strategies have been used in zebrafish for both forward5 and reverse genetic screening6. Comprehensive genetic mapping and annotation in zebrafish have facilitated gene-editing approaches as a primary technique to engineer targeted gene knockouts (KOs) and precise knock-ins (KIs)7.
Despite this, generating precise KI edits in zebrafish is limited by low efficiencies and the difficulty of accurate detection. Although transcription factor-like effector nucleases (TALENs) have been successfully used and optimized for KIs8, CRISPR provides an improved gene-editing strategy with simpler sgRNA targeting. Numerous studies have used CRISPR to generate precise KIs in zebrafish9,10,11,12,13,14,15,16,17,18,19,20, although these edits generated through CRISPR-mediated homology-directed repair (HDR) tend to be inefficient with low intrinsic success rates that require genotyping as a primary screen9,10,14,21. This demonstrates the need for an efficient KI CRISPR system in zebrafish, as well as a reliable high-throughput system for detecting precise edits.
The goal of this study was to describe a platform for generating a precise cardiac gene KI in zebrafish hearts with simple and high-throughput detection of successful edits. A CRISPR-Cas9-based two-sgRNA exon replacement approach is described, which is based on a TALEN approach8. This approach involves excision of the target sequence using two-sgRNA guides and replacement with an exogenous template sequence that contains the KI of interest as well as a genetically encoded intronic reporter gene (Figure 1). The integration of a genetically encoded fluorescent reporter within the target gene intronic sequence enables the efficient detection of positive edits. A phenotyping platform is then described for assessing cardiac electrical function in zebrafish larvae for non-invasive characterization of the gene variants associated with inherited LQTS, a cardiac electrical disorder that predisposes individuals to sudden cardiac death.
These approaches will enhance the access to and use of zebrafish KI gene edits to model inherited diseases and address biological and physiological questions, such as mapping gene expression patterns, and developmental regulation. Since zebrafish hearts better parallel human cardiac electrophysiological characteristics than murine models, they may be particularly attractive as a genetically tractable system for cardiac disease modeling7,22,23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Program</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CRISPOR</td>
			<td>TEFOR Infrastructure</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ENSEMBL</td>
			<td>European Bioinformatics Institute</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health (NIH)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Manager</td>
			<td>Open Source (Github)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NEBiocalculator</td>
			<td>New England Biolabs (NEB)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Plate</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm Petri Dish</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blackfly USB3 Camera</td>
			<td>Teledyne FLIR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415C</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZNA Gel Extraction Kit</td>
			<td>Omega Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MAXIscript T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQ 5000 Incubator</td>
			<td>Barnstead Lab Line</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep Kit</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mMessage mMachine T7 Ultra Transcription Kit</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ND1000 Spectrophotometer</td>
			<td>Nanodrop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Purification Kit</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLI 100A Picoinjector</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 305 Steroscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Mini Sub Cell GT Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZebTec Zebrafish Housing System</td>
			<td>Tecniplast</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SERVICES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Synthesis</td>
			<td>Genewiz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sanger Sequencing</td>
			<td>Genewiz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10&#946; Competent Cells</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X PCR Buffer</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mM Nucleotide Mixture</td>
			<td>ABM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI Endonuclease w/ buffer</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BsaI Endonuclease w/ buffer</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DR274 Plasmid (XL1 Blue bacterial agar stab)</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI Endonuclease w/ buffer</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HindIII Endonuclease w/ buffer</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Blue</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MLM3613 Plasmid (XL1 Blue bacterial agar stab)</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MS-222 (Tricaine)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pKHR5 Plasmid (DH5&#945; bacterial agar stab)</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PmeI Endonuclease w/ buffer</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SalI Endonuclease w/ buffer</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Ligase w/ buffer</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taq Polymerase</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Hydrochloride</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI Endonuclease w/ buffer</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RECIPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solution</td>
			<td>Component</td>
			<td>Supplier</td>
			<td></td>
		</tr>
		<tr>
			<td>Annealing Buffer (pH 7.5-8.0)</td>
			<td>10 mM Tris</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>50 mM NaCl</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 mM EDTA</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>E3 Media (pH 7.2)</td>
			<td>5 mM NaCl</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.17 mM KCl</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.33 mM CaCl2</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.33 mM MgSO4</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Buffer (pH 7.5)</td>
			<td>20 mM HEPES</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>150 mM KCl</td>
			<td>Sigma</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr-cas9-mediated precise knock-in edits in zebrafish hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

The successful use of this two-sgRNA exon replacement CRISPR approach is highlighted by the introduction and simple detection of a precise edit to engineer the LQTS-associated variant, R56Q, in the zkcnh6a gene in zebrafish. Figure 6 shows a representative 3 dpf larvae injected at the one-cell embryo stage with CRISPR components as described above. Figure 6A shows the presence of the YFP mVenus reporter gene expression in the eye lens as a positive repo...

Watch this Scientific Journal Video about CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts at JoVE.com

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

Our protocol provides a pipeline for both precise integration and simple visual detection of CRISPR edits, as well as an example of post-edit phenotyping. The main advantage of this approach is ease of CRISPR edit detection, allowing for efficient and simplified identification of successful edits. This approach allows for simple generation and characterization of disease-associated variants in zebrafish.Here, we are examining long QT syndrome. Follow-up studies on pharmaceutical therapies are then feasible to explore. Begin by designing two sgRNA guides to excise the knock-in target site sequence by identifying the zebrafish ortholog for the gene of interest.Use Ensembl to locate the target site within the gene sequence of interest, including the two kilobase flanking sequence used to make the template. Use a design software tool such as CRISPOR and select Danio rerio species and Cas enzyme. For the two sgRNA approach, choose one sgRNA before the target exon and a second sgRNA within the immediate downstream intron.Ensure that the selected sgRNAs have high specificity and low predicted off-target binding. Use CRISPOR rankings to identify guides with minimal off-target binding. Now identify the most likely potential off-target sites for PCR-based Sanger sequencing genotyping.After selecting the two sgRNAs, obtain the reverse complement for each, two complementary oligos that precede the mutation and two complementary oligos downstream of the mutation. Add compatible restriction sites on each oligo to incorporate the guide in the plasmid of choice. For the integration of the selected guide sequences into the DR274 plasmid, create an overhang using a five prime BSA1 restriction site and engineer the BSA1 recognition sites at the five prime ends of the guides.Next, design the exogenous template for HDR in zebrafish by choosing two sequence fragments that will flank M venous YFP reporter gene housed in the PKHR5 plasmid. Divide the template into two segments to insert it on either side of the M venous YFP reporter gene. Ensure that the split site is in an intron to avoid cutting the coding sequence.Incorporate all the required modifications mentioned in the manuscript to facilitate cloning into the PKHR5 plasmid. Inject the embryos in the one cell stage at approximately 40 minutes post-fertilization. After three days of fertilization, anesthetize zebrafish larva by transferring it to a 25 millimeter Petri dish containing 0.3%MS222 until they lose their self-righting reflex.Once anesthetized, transfer the larva into the well of a 24-well plate. Using a microscope capable of detecting GFP or YFP, screen for reporter gene fluorescence in the eyes of the larva. After capturing the images of the larva, document the presence or absence of the reporter gene expression.To confirm accurate and precise HDR gene editing, perform genotyping by first anesthetizing the larva three days post-fertilization as demonstrated earlier and isolating the genomic DNA from the tail clip using the hotshot method. Add the excised tail clip to 15 microliters of 25 millimolar sodium hydroxide and incubate it 95 degrees Celsius for 20 minutes, then neutralize with 1.5 microliters of tris hydrochloric acid. Centrifuge at 13, 800 times G for 30 seconds, and retain the supernatant containing the extracted genomic DNA.Recover the larva in E3 media and return it to the housing system if further study is intended. Using the extracted genomic DNA as a template, perform PCR-based Sanger sequencing of on-target and potential off-target sites. Ensure the on-target primer design captures the mutation site and the closest sgRNA binding site.Design a separate sequencing primer to detect the transition from the inserted homology arm and the target gene to confirm integration into the gene of interest. Design primers to sequence the top three potential off-target sites. Compile on and off-target genotyping, heart rate, pericardial dimensions, ECG phenotyping, and reporter gene data identifiable for each zebrafish.The YFP M venous reporter gene expression was observed in the islands, acting as a positive reporter of successful template integration. Reporter gene positive fish were found to have the precise edit G2A which introduces the R56Q variant into ZKCNH6A. The measurement of pericardial dimensions as a ratio of the eye area is shown here.A trend of bradycardia with increasing pericardial edema associated with the disorders of cardiac repolarization in zebrafishes was observed in the R56Q gene edited larvae. ECG recording from a zebrafish larva heart three days post-fertilization is shown here. The most important aspect of this approach is guide design, as is often the case in CRISPR procedures.Effective guides are essential for successful CRISPR edits. Alternative CRISPR methods can be followed allowing for more precision or other functionalities. Additionally, large genes can be inserted such as a prime editing complex providing an in vivo inducible editing system.

crispr-cas9-mediated-precise-knock-in-edits-in-zebrafish-hearts

Research

JoVE Journal

Biology

3.1K visualizações.  Simon Fraser University. Nosso protocolo fornece um pipeline para integração precisa e detecção visual simples de edições CRISPR, bem como um exemplo de fenotipagem pós-edição. A principal vantagem dessa abordagem é a facilidade de detecção de edição CRISPR, permitindo a identificação eficiente e simplificada de edições bem-sucedidas. Esta abordagem permite a geração e caracterização simples de variantes associadas à doença em peixes-zebra.Aqui, estamos examinando a síndrome do QT long...