The overall goal of this HMEJ strategy is to provide a promising genetic tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies. This HMEJ passed the measure. Can have also key questions in the genetic and in neuro field.
Such as how to improve the efficiency of precise targeted integration of transgenes in vivo. The main advantage of this HMEJ based strategy is ability to correct gene mutations with high efficiency in vivo. Which followed is therapeutic in potential.
Demonstrating the procedure will be Xing Wang, a graduate student from my laboratory. To begin the protocol, collect 5000 N2a cells, previously transfected with Cas9 sgRNA EGFP expression vectors by fluorescence activated cell sorting or FACS, using non-transfected cells as a control. Digest the collected cells in 2 to 5 microliters of lysis buffer at 56 degrees Celsius for 30 minutes.
Then heat and activate the proteinase K at 95 degrees Celsius for 10 minutes. Amplify the sample by nested PCR using the manufacturer's protocol. Mix one microliter of the lysis product with DNA polymerase and a pair of outer primers recognizing the sequence around the sgRNA target site and perform the primary PCR in a total volume of 20 microliters.
Next, activate DNA polymerase at 95 degrees Celsius for 5 minutes, then perform the primary PCR with a final extension at 72 degrees Celsius for 5 minutes. Perform the secondary PCR using one microliter of primary PCR product and a pair of nested inner primers. Denature and re-anneal 300-600 nanograms of purified PCR product in 20 microliters of 1x TC7EI reaction buffer.
Add one microliter of T7EI enzyme to the annealed PCR products and digest them at 37 degrees Celsius for two hours. Then run the digestion product on 2%agarose gel and 1x TAE buffer at 120 volts for 40 minutes, until the fragments are separated. Use ImageJ to determine the band intensities of cut and uncut DNA and calculate the indel frequency.
For Cas9 mRNA preparation, add the T7 promoter sequence to the Cas9 coding region by PCR amplification. Next, add the primer Cas9 FR at a final concentration of 0.1 micromolar and 20 nanograms of Cas9 expressing vector to the 1x high fidelity DNA polymerase mix. Adjust the final volume to 50 microliters with nuclease free water.
Activate DNA polymerase at 95 degrees Celsius for 5 minutes and perform the PCR. Purify the T7 Cas9 and PCR product for in vitro transcription, or IVT. Then transcribe 0.5 to 1 microgram of DNA, using an mRNA transcription kit at 37 degrees Celsius for 8 hours in a total volume of 20 microliters.
Add 1 microliter of DNase to the mixture to remove the DNA template at 37 degrees Celsius for 15 minutes. After adding a poly(A)tail for 45 minutes at 37 degrees Celsius, recover the Cas9 mRNA with an RNA purification kit. Generate the sgRNA template driven by a T7 promoter with high fidelity DNA polymerase as performed in the previous section and choose an sgRNA scaffold containing vector as the template.
After purifying the T7 sgRNA PCR product, use 0.5 to 1 microgram of DNA as the template for in vitro transcription of sgRNA using a short RNA transcription kit at 37 degrees Celsius for 6 hours, in a total volume of 20 microliters. After 6 hours of incubation, add 1 microliter of DNase to the mixture and continue the incubation at 37 degrees Celsius for 15 minutes to remove the DNA template. Purify sgRNAs again using the RNA purification kit.
Dilute the sgRNA to 500 nanograms per microliter in RNase free water and store the samples at minus 80 degrees Celsius for up to three months. High quality of Cas9 mRNA and sgRNA with no degeneration and the correct line of HMEJ donor parameters are the most critical steps in the generation of knock-in mice. Place the fertilized embryos into KSOM medium at 37 degrees Celsius in an incubator with 5%carbon dioxide.
Mix 100 nanograms per microliter of Cas9 mRNA, 50 nanograms per microliter of sgRNA, and 100 nanograms per microliter of HMEJ donor vector and add water to adjust the final volume to 10 microliters. Pipette the mixture up and down and place it on ice. Next, pull capillary needles using a micropipette puller.
Inject a probable volume of the mixture into the cytoplasm of zygotes with well defined pronuclei in a droplet of HEPES-CZB medium containing 5 micrograms per milliliter Cytochalasin B using a microinjector with constant flow settings. Finally, culture the injected zygotes in KSOM medium at 37 degrees Celsius under 5%carbon dioxide until the blastocyst stage after 3 1/2 days for fluorescence observation. After 3 days of injections, 12.9%of the blastocysts receiving HMEJ donors were positive for mCherry, which was strictly expressed in the trophectoderm.
By sequencing the PCR positive mice, all examined integration events were precise in-frame integrations at both 5 prime and 3 prime junctions. After 7 days of injections, immunofluorescent images revealed that nearly half of the transfected hepatocytes expressed mCherry as stained on the liver sections. After the withdrawal of NTBC, immunohistochemistry liver staining of Fah corrected hepatocytes of Fah negative mice receiving Fah HMEJ and Cas9 constructs showed more effective proliferation than MMEJ based method.
The HMEJ based strategy could be an ideal platform for replacing mutated sequence such as part of mutation with the correct one. Which is not applicable for an HHEJ based method. After this experiment, this therapy paves the way for researchers into field where precise target integration to explore broad therapy applications.
