CRISPR / Cas9 Mediated Genome Engineering Services

Introduction

The service includes:
Purchase of egg donors.
Hyperovulation of egg donors and mating with stud males.
Production of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the morning of injection.
Harvesting of eggs from euthanized donors.
Dilution of the client’s ssODN or circular plasmid DNA with injection buffer, combination with tracrRNA and crRNA, plus Cas9 protein, and microinjection (or electroporation) of this mixture into one pronucleus of each of at least 120 fertilized eggs.
Transfer of all eggs that survive the injection process into the oviducts of pseudopregnant foster mothers.
Monitoring of foster mothers before and after birth of pups.
Marking pups in each litter by distal toe-clip, sexing, and tail-cutting at about 10 days of age.
Transfer of tail biopsies to the client for genotyping (or in house genotyping for the client for an additional fee).
Weaning, identification, and transfer or shipment of transgenic pups to the client.
Breeding of G0 founder animals and genotyping of G1 animals can be performed for an additional fee.

Summary

The strain with the highest yields is the hybrid strain, B6SJLF1/J. Egg donors and stud males are purchased from the Jackson Lab (Jax), where they are produced by mating C57BL/6J females with SJL/J males. When B6SJLF1/J males and females are mated in our facility to produce fertilized eggs for microinjection, the eggs and resulting pups are referred to as B6SJLF2. B6SJLF1/J mice are genetically identical - each pair of chromosomes consists of one C57BL/6J chromosome and one SJL/J chromosome. In contrast, their B6SJLF2 offspring, while still being approximately 50% C57BL/6J and 50% SJL/J, are not genetically identical due to meiotic recombination, and will have a variety of coat colors.

Service Features

The standard Targeted Transgenesis service includes the following:
Linearization and purification of the targeting construct DNA.
Electroporation into ES cells.
Growth of cells under selection.
Picking at least 24 drug-resistant colonies into a 96-well plate.
Splitting the colonies into duplicate plates.
Freezing one plate and extracting DNA from the other plate.
Digestion of ES cell colony DNA with restriction enzyme.
Agarose gel electrophoresis and gel photography.
Capillary transfer of digested DNAs to nylon membrane.
Hybridization of membrane with radioactive probe (standard ROSA26 probe supplied by the TMF).
Washing blot and imaging on phosphorimager.
Analysis and annotation of image.

Analysis Methods

We prefer to microinject or electroporate a pre-assembled Cas9/crRNA/tracrRNA ribonucleoprotein complex, which can reduce mosaicism.
We have generated mutants using Cas9/crRNA/tracrRNA in both inbred (e.g. C57BL/6NJ, C57BL/6J, FVB/N) and hybrid (B6SJLF2) embryos. Use of hybrid embryos (and electroporation) usually produces significantly larger numbers of mice for subsequent analysis. If your desired modification might be obtained at a low frequency, we encourage you to consider whether you can tolerate generating this in a hybrid strain background.
During planning, we identify gRNA sequences that minimize likelihood of off-target effects on the same chromosome. Unlike working with mammalian cells, breeding mice allows segregation of any unlinked off-target effects.
In our experience it is vital to identify gRNA that cut as close as possible (<10 bases) of where the desired genetic modification is to be introduced.

Turnaround Times

The minimum time needed to go from electroporation of the targeting construct to clone DNA ready for genotyping is about 4 weeks. The actual time may be somewhat longer if other targeting jobs are already in progress.
Southern blotting turnaround times can vary, depending on how many projects are in progress. At present, the minumum time is about 10 days.
Clone expansion and DNA extraction require an additional 2 weeks.

Advantages

This service offers the following advantages over the traditional method of pronuclear injection:
Similar levels of expression in all founder lines.
A known locus of integration.
Transgenes are always inserted as a single copy, minimizing the chance that they will be silenced by methylation or lost via recombination.
As the site of integraton is known, only a single line of "transgenic" mice is required.

CD Genomics has already updated its technology platforms to mainstream NGS, long-read sequencing, and microarray instruments. We continue to strive to offer the same reliable services to pharmaceutical and biotechnology companies, as well as academia and government agencies, for all your sequencing or array needs. If you are interested, please contact us directly for assistance.