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

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.