Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout Mice Generation

The scope of our research is to simplify the generation of knockout mice by using in utero electroporation, delivering genome editing components directly into the oviducts of pregnant females. Micro-injections into zygotes requires maintaining a large number of animals including donor and recipient females and vasectomized males. Therefore, protocols that minimize animal use are preferable.

The use of Nepa21 system for zygote electroporation provides a unique opportunity to generate targeted gene knockout mice in a single step, with efficiency comparable to standard nuclear injection-based techniques To begin, use computational analysis tools to identify potential single guide RNA sequences targeting the selected gene of interest. Select sequences located in exons that are as close as possible to the desired mutation site. Use a genome browser to cross-reference exome structure and prioritize sequences present in most splice isoforms.

Analyze the predicted off-target activity of each guide sequence using the tool's integrated scoring system. Choose sequences that demonstrate the lowest likelihood of off-target binding. To produce a linear double-stranded DNA template, prepare the PCR master mix using the components specified in the given table.

Add the polymerase last and keep the master mix on ice to preserve enzymatic stability. Then, run the PCR using the following thermal cycling conditions. Next, prepare the in vitro transcription master mix using the given components and incubate the reaction for 1.5 hours at 37 degrees Celsius using a thermocycler for consistent temperature control.

Then, add two microliters of DNase I to the reaction. Incubate at 37 degrees Celsius for an additional 30 minutes to digest the DNA template, and then inactivate the enzyme at 75 degrees Celsius for five minutes. After TRIzol purification of RNA, measure the RNA concentration using a spectrophotometer and check the A260 to A280 and A260 to A230 ratios.

To prepare the ribonucleoprotein complex for transfection, dilute one molar Tris-HCl buffer to a concentration of 100 millimolar using nuclease-free water. Filter the solution using a syringe filter with a 0.022 micrometer pore size. Aliquot into 10 microliter portions and store at four degrees Celsius until use.

Next, prepare the transfection mix using spCas9 nuclease, single guide RNA, and Tris-HCl diluted with DEPC water in a nuclease-free tube. Incubate the transfection mix at 37 degrees Celsius for 10 minutes to allow formation of the ribonucleoprotein complex. Then, aliquot the transfection mix into small volumes suitable for single I-GONAD sessions and keep it at four degrees Celsius to preserve reagent activity.

After mating CD1 female mice with single-housed CD1 male mice overnight, check each female for the presence of a copulation plug. Transfer only those females with visible plugs into a new cage for further experimental procedures. Autoclave all stainless steel surgical instruments to ensure sterility before use.

Then, weigh the selected plug=positive female mice using an electronic scale before anesthetizing them. Monitor the anesthetized mice for loss of the toe pinch reflex to confirm adequate anesthesia. Place the mouse on a heating pad to maintain body temperature during the procedure.

Then, apply veterinary ointment to the eyes to prevent dryness. Using fine scissors, make a 0.7 centimeter longitudinal incision in the skin of the lower back region. Then, laterally spread the slightly open tips of the scissors to gently extend the skin incision to approximately 1.3 centimeters.

After that, carefully make a 0.7 centimeter incision in the peritoneum to expose the abdominal cavity. Extend the peritoneal incision to 1.1 centimeters, bilaterally pushing with slightly open scissor tips. Locate the fat pad connected to the ovary and gently pull it outward using forceps until the ovary, oviduct, and uterus are exposed.

Using a vessel clamp, secure the fat pad to stabilize the reproductive structures. Next, load two microliters of the ribonucleoprotein injection mix into a disposable plastic capillary using a stripper pipetter. Using micro scissors, make a 0.2 centimeter incision in the oviduct wall a few millimeters upstream of the ampulla.

Insert the plastic capillary attached to the stripper pipetter into the oviduct incision and carefully expel the injection mix without introducing air. Gently withdraw the plastic capillary from the oviduct to complete the injection. Turn on the electroporator by pressing the power switch on the front panel.

Set the electroporation parameters on the device as shown on the screen. Using tweezer-type electrodes, gently grasp the oviduct and press the Kiloohm button to measure the resistance. Immediately after resistance measurement, press the Start button to begin electroporation and wait for the end signal to appear on the Ohm button.

After electroporation of the first oviduct, gently return the reproductive tract to the abdominal cavity. Close the incision using 4-0 polyglycolic acid surgical suture. For genotyping, design 18 to 21 nucleotide long primers with a melting temperature near 60 degrees Celsius to generate a 300 to 600 base pair PCR product.

To confirm the specificity of each primer, perform a BLAST search against the mouse genome database to eliminate the risk of non-specific amplification. Next, for DNA extraction, using an ear punch, collect a small tissue sample approximately two millimeters in size from the mouse's ear. Add 100 microliters of lysis buffer.

Incubate the sample at 95 degrees Celsius for 60 minutes, vortexing every 20 minutes to ensure efficient lysis. After performing Sanger sequencing, analyze the results to assess genome editing outcomes. To use ICE analysis, upload both the Sanger sequencing files, enter the guide RNA sequence, and select the appropriate nuclease used in the CRISPR experiment.

Next, to use TIDE, upload the control and experimental sequencing files in AB1 format and enter the guide RNA sequence. ICE analysis detected three distinct indel variants at the Cas9 cut site, a one base pair deletion in 42%of sequences, a three base pair deletion in 25%of sequences, and a 23 base pair deletion in 27%of sequences. The model fit value was 0.94 and the knockout score was 69, indicating a strong predicted correlation and likely gene knockout.

Sequence chromatograms confirmed disruption at the Cas9 cut site in the edited sample, whereas the control chromatogram showed an intact sequence at the same site. Out of six electroporated mice, only four produced pups with mutations, resulting in a 60%overall editing efficiency. All litters across the six electroporated mice showed reduced pup numbers, with only three to four pups per mouse.