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

CRISPR-based genome editing tools have greatly facilitated the production of genetically engineered rep models. The CRISPR system is comprised of a single guide RNA complex to a Cas9 nuclease and can be programmed to target a sequence of interest within the genome to create a double stranded DNA break. Through the codelivery of a DNA repair template, this DNA break can be precisely restored along with the addition of any desired engineered DNA sequence through a process called Homology Directed Repair.

It is by this process that we are able to generate knock-in rat models with targeted DNA insertions or substitutions. Using this protocol, we present a modified approach using adeno-associated virus to deliver a DNA repair template to rat embryos, along with a subsequent delivery of CRISPR Cas9 complexes through two cell embryo electroporation. AAV serotypes 1 or 6 is packaged with an engineered DNA repair template designed to be integrated into the rat genome through the CRISPR-mediated Homology Directed Repair process.

The AAV is added to a 30 microliter drop of rat KSOM media at a final concentration of 3 X 10 to the 7th genome copies per microliter. Next, freshly collected one cell stage embryos with visible pronuclei are then transferred to the media containing AAV allowing for viral transduction to occur. Using our protocol, there is no need to thin the zona pellucida as AAV serotypes 1 and 6 are able to readily pass through for efficient DNA repair template delivery.

Embryos are cultured with the AAV overnight at 37 degrees Celsius with 5%carbon dioxide and maximum humidity. The following day, an electroporation mixture is made containing a hundred nanograms per microliter of single guide RNA and a hundred nanograms per microliter of Cas9 protein diluted in OptiMIM media. The mixture is incubated at room temperature for 10 minutes to allow for formation of CRISPR RNP complexes.

During the incubation time, the electroporator is turned on and leads are attached to the glass slide electrodes. After the RNP formation is complete, five microliters of electroporation mixture is pipetted between the electrodes, taking care not to introduce air bubbles to the solution. Embryos, now at the two cell stage, are removed from the media containing AAV and carefully moved to the electroporation mixture between the electrodes.

Embryos are aligned, ensuring that none are touching either electrode as this will cause lysis during the electroporation. Here is a view of this procedure through the microscope. It is also important to note that impedance will decrease as the volume between the electrodes increases.

For this reason, and as to not dilute the electroporation mixture, minimal media should be transferred along with the embryos. Next, the impedance is measured by pressing the button with an ohm symbol on it. The impedance should be within the range of 0.18 to 0.3 ohms.

If in range, the electroporation is started by pressing the start button. This process only takes a couple of seconds, and this is what it looks like through the microscope. You will notice bubbles quickly forming on each electrode.

Immediately after the electroporation, embryos are collected from the glass slide and washed three times in fresh rat KSOM media. The embryos can then be cultured for in vitro studies or transferred to surrogate females to produce live offspring. It is important to note that embryo swelling is common after electroporation.

This swelling should subside after a few hours on culture. It is also common to see cellular fusion events in up to 20%of two cell embryos after electroporation. Cellular fusion can typically be avoided by careful horizontal axis alignment of the embryos between the electrodes.

Testing our protocol in rat embryos and screening cultured blasts for an insertion of an engineered short artificial intron sequence containing two loxP sites. We noted over 60%of blastocysts contain the correctly targeted knock-in allele based on next generation sequence analysis. Next, further testing our protocol to produce live knock-in rats, we design a project to engineer a Cre recombinase coating sequence targeted to the ATG start site of the rat DRD2 gene.

Of the 11 offspring born, 10 were positive by PCR analysis across the five prime homology arm, three prime homology arm, and an internal PCR assay to detect Cre recombinase. As a summary over seven different projects, we have achieved a 76%average knock-in success rate in founder animals as determined by PCR analysis across homology arm junctions and DNA sequence verification. Using this AAV mediated DNA delivery and two cell embryo electroporation pipeline, we have experienced significantly higher knock-in efficiencies than traditional approaches for DNA insertions up to three KB in length.

It is also important to note that while we demonstrated the use of this procedure in rat embryos, it may be effectively applied to other species for generating targeted DNA insertions as well.