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Cas9/gRNA Mediated Genome Engineering Services

microinjection of fertilized mouse oocytes

Background

As with many other species, genome engineering in the mouse has been revolutionized by use of the CRISPR / Cas9 system. As of August 2017, the TMF has successfully conducted 38 independent projects to modify the mouse genome via use of Cas9/gRNA, with 11 more currently in progress. Most completed projects have involved introduction of point mutations (e.g. to generate amino acid changes), loxP sites and exon deletions via Cas9/gRNA in combination with single-stranded oligodeoxynucleotide (ssODN) as a template for homology dependent repair (HDR). We have also had success using double-stranded plasmid based templates for repair, although the frequency of successful targeting is lower than projects using ssODN as the template for HDR. Insertion of relatively large sequences of DNA (i.e. up to 2 kb in length) can be accomplished using IDT's MegaMers (essentially, very long single stranded and modified DNA) and to date we have modified four independent loci by inserting sequences encoding fluorescent proteins (i.e. mRFP, EGFP, mVenus etc) using this approach. This technology is evolving at a rapid pace and we continue to evaluate modifications of methods that may significantly improve efficiency, especially targeting of large DNA insertions, while using fewer animals.

We strongly encourage investigators interested in modifying the mouse genome to contact Jon Neumann, Managing Director of the TMF, before ordering any reagents to perform Cas9-genome modification in the mouse. We can help you overcome commonly experienced pitfalls that arise during the design phase while planning genome modification in mice using Cas9/gRNA based methods.

Please note, all mouse genome modification performed by the TMF involves microinjection of Cas9 / gRNA plus ssODN or other repair template into fertilized mouse oocytes. The TMF currently does not currently offer services to perform genome engineering in mammalian cell lines.

We prefer investigators first contact us to discuss the genome modification they would like to engineer in the mouse. We will evaluate the locus involved, and provide a strategy for its modification. We are also willing to perform microinjection of ssODN, gRNA etc purchased from approved commercial vendors (e.g. IDT) after we have evaluated the design strategy and materials involved, including experimental evidence that they will be productive. For questions and further information, contact Jon Neumann.

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Some comments about how we plan and perform genome modification using Cas9/gRNA

  • We have had success using single-guide (sg) RNA's as well as mixtures of gRNA and tracrRNA. Our current preferred method is to purchase tracrRNA and gRNA that have modified bases (Alt-RTM) from Integrated DNA Technologies (IDT).  These have low toxicity and higher stability in mammalian cells.
  • We have had success microinjecting mRNA that has a relatively long polyA tail encoding Cas9. We have also had success using commercially sourced Cas9 protein. However, in our experience, not all commercial sources of Cas9 have similar activity and quality. Microinjecting a pre-assembled Cas9/gRNA/tracrRNA ribonucleoprotein complex may reduce mosaicism, but can also reduce the number of founder animals with the desired genome modification.
  • We have generated mutants using Cas9/gRNA in both inbred C57BL/6NJ and hybrid B6SJLF2 embryos. Use of hybrid embryos almost always 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.
  • We try to 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 (within a few bases) of where the desired genetic modification is to be introduced.
  • To introduce genetic changes with a HDR template, we routinely use ssODN (Ultramers from IDT, not PAGE purified) that have 30 - 60 base "arms" flanking the cut site. To introduce longer sequences of DNA (up to 2 kb) we routinely use IDT's MegaMers, with ~ 60 - 100 bases of flanking sequence homologous to the target site.
  • More than one DNA mutation can be introduced concurrently using longer (i.e. up to ~ 170 bases) ssODNs. However, the likelihood that the desired mutations will be incorporated decreases with increasing distance from the cut site.
  • Whenever possible, we routinely test at least 3 independent gRNA's at each site where cutting is desired, especially for sites in introns.  High efficiency of cleavage usually correlates with efficient genome modification. For sites in exons, we usually design 2 gRNA for each site and inject without testing. Typically one of the sites works better than the other even when both have similar predictive scores.
  • We design the ssODN's or plasmid template to include DNA polymorphisms that prevent re-targeting of repaired sequence by Cas9/gRNA. These can be introduced in the PAM sequence or in the seed sequence proximal to the PAM.
  • Because embryos or mice can be homozygous for a targeted event, we screen DNA from tests of gRNA and founder (G0) mice using fragment analysis or sequencing, in addition to T7 endonuclease mismatch assay.
  • Founder mice should be expected to be mosaic for genetic modifications (the TMF has obtained G0 mice that transmitted at least 5 independent alleles at an autosomal locus). This may happen as a result of persistent Cas9/gRNA activity in the blastomeres of the pre-implantation (i.e. 4 cell, 8-cell, 16 cell stage etc) embryo. Thus screening methods should be capable of detecting the varied outcomes that occur with CRISPR.
  • We try to identify founder (G0) generation mice for breeding that have evidence by PCR or Southern analysis of at least 30% of the signal being the desired genetic change. Rather than perform Next-Gen sequencing of DNA from G0 animals, we have found it more productive to breed the G0 with a wild type mouse and validate the modified allele in the G1 generation. This backcross has the advantage of starting the process of removing any unlinked mutations that may have arisen in the G0 generation.
  • To date, all Crispr projects in the TMF have utilized Cas9 from S. pyogenes. The use of other varieties of enzymes would be considered, but require additional planning and costs to acquire the necessary reagents. 

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Ordering

Contact Jon Neumann to discuss pricing options and to request a customized Service Request Form (SRF). The completed SRF must be signed by the Principal Investigator.  Signed SRF's can be delivered to Jon Neumann via mail, fax, or as a scanned or photographed image.

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Performance Guarantees 

The TMF has a high success rate with Cas9-mediated genome modification. 38 / 46 projects have been successful, with the unsuccessful projects mostly being early efforts to introduce loxP sites concurrently or to introduce large pieces of DNA via sub-optimal plasmid HDR templates. Lessons learned during these early failures have been applied to more recent genome modification attempts. We feel confident that when we are consulted before the design phase and when we develop the materials with which to perform the genetic modification, we can predict whether there will be a high likelihood of success. However, the combination of the locus to be modified plus the type of modification required greatly influences the likelihood of success. For this reason we do not currently offer a service guarantee to produce animals with the desired genetic modification.

For investigators that send us materials for microinjection, the only service guarantee provided is that we will inject a minimum of 120 fertilized oocytes and transfer any surviving oocytes to pseudo-pregnant recipient females. Note, all our experience in oocyte injection has been with RNA and protein for Cas9 and gRNA. The TMF discourages use of plasmid DNA-based introduction of Cas9/sgRNA.

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Service Description

The service includes:

  • Purchase of egg donors.
  • Superovulation 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 gRNA, plus Cas9 mRNA and protein, and microinjection 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.
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Which Mouse Strain Should I Use ?

We routinely offer 2 strains of egg donors. The service fee varies by strain because of differences in egg and pup yields among these strains.

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.

The C57BL/6NJ strain, from Jax, is our more expensive strain because it produces the lowest egg and pup yields. However, its rate of success in producing modified animals (percentage of pups that have the desired modification) is about the same as the other strains we routinely offer.

While other strains can be used as egg donors, these may require additional fees. Some lines are very poor donors and should be avoided if possible (e.g., BALB/c and DBA/2J). 

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