The Beth Israel Deaconess Transgenic Core Facility opened in October of 1991.The Transgenic Core is a state-of-art facility that produces genetically altered animals.
New development in CRISPR-related mouse genetic engineering services offered by the BIDMC Transgenic Core (updated: 07/19/2017)
Using “Easi-CRISPR” (Quadros RM, Genome Biology, 2017, PMID: 28511701), we can now rapidly and easily generate Cre knockin mice, as well as many other types of mice, via one-step zygote injection of gene targeting reagents. Importantly, the homologous recombination process is extremely efficient, even for targeted insertion of large fragments like Cre. This approach bypasses the need to do gene targeting in mouse embryonic stem cells, greatly reducing the work and time needed to create gene targeted mice.
The key advances of “Easi-CRISPR” are: a) use of single-stranded DNA (ssDNA) targeting constructs which greatly increases targeting efficiency, and b) the ability of a commercial supplier to now synthesize long stretches of ssDNA allowing for targeted insertion of large DNA fragments (up to 2 kb, which includes Cre, GFP, a loxed exon, etc.).
The method is simple and requires little or no molecular biology skills; hence is accessible to all labs. The steps are essentially as described below:
Step 1: You select a sgRNA which will direct Cas9 to cut genomic DNA at or near the desired site of targeted insertion.
Step 2: You design, in silico, your targeting construct sequence. To make a knockin p2A-Cre driver mouse, this construct would be ~1,300 bases long and from 5’ to 3’ would include ~100 bases of target gene 5’ homology arm sequence, 66 bases of p2A sequence, ~1,032 bases of Cre sequence, and ~100 bases of target gene 3’ homology arm sequence.
Step 3: A company then synthesizes your “in silico” targeting construct as ssDNA at $0.90/base. See link below:
Step 4: The BIDMC Transgenic Core then injects a mixture of your sgRNA, your ssDNA targeting construct and Cas9 protein (which you can purchase) into fertilized zygotes. For BIDMC labs, the cost of this injection is currently $3,500 for FVB zygotes and $5,000 for C57BL/6 zygotes. For academic labs located outside BIDMC, costs are $5,250 and $7,500, respectively.
Step 5: About 4 weeks later you will receive tails from the live-born F0 mice. You then isolate genomic DNA and perform PCR across the 5’ and 3’ arms to detect the F0 founder mice with targeted knockin.
Step 6: Identified knockin F0 founders are then ready to breed for further use.
The Quadros reference cited above reports very high efficiency for Easi-CRISPR on many different targets. We have just completed a test of this approach to generate p2A-Cre knockin mice. 12 of 24 live-born F0 mice had p2A-Cre inserted homologously into the endogenous gene. And one of these 12 targeted mice was homozygous for the p2A-Cre knockin allele. Clearly, as reported by Quadros et al., the method is extremely efficient. And importantly, it is extremely simple to do – requires no molecular biology skills. Finally, because the 5’ and 3’ homology arms are very short (~100bp), the targeted allele is extremely easy to genotype using short-range PCR.
As mentioned above, the approach can be used to insert Cre, but also many other things including Flp, Dre, XFP, an epitope tag, etc. Also, it can be used to place lox sites around an exon of a gene.
If you are interested in using this approach to generate mice, feel free to contact me (firstname.lastname@example.org) and/or Chen Wu (email@example.com). Chen Wu is the resident expert in my group on using CRISPR to engineer mice. Chen can provide detailed advice and guidance on any of the steps mentioned above. Joel Lawitts (firstname.lastname@example.org) directs the Transgenic Injection Facility at BIDMC and can answer questions related to “Step 4”.
FVB/N or C57BL/6 CRISPR injections
Service: Pronuclear stage zygotes are injected with CRISPR reagents for the purpose of creating knockouts or knockins. We have observed increased efficiency when investigators have purchased all their reagents (Cas9 enzyme, nickase, sgRNA and DNA). In 3 recent (October ’15) knockin projects, utilizing Cas9 nickase and two sgRNA9(s) there were 5/65, 9/73 and 6/59 (founders/pups screened). The source of reagents were:
- Recombinant Cas9 nickase: PNA bio Inc (www.pnabio.com); catalog number CN01; 50ug/vial. The company also provides Cas9 enzyme (see website for cat #).
- sgRNA(s): PNA Bio Inc (www.pnabio.com); (sgRNA synthesis service; 50ug lyophilized). No catalog number.
- Oligo DNA (knockins): IDT (www.idtdna.com) as a 4 nmole Ultramer DNA oligo.
The reagents are injected in the following concentrations (manufacturer recommendation): Cas 9 (200ng/ul), sgRNA(s) (100ng/ul), DNA (20-60ng/ul). A mix of the reagents totaling 50ul is made and is used for one week. Reagents may be presented to the Core in a lyophilized form or reconstituted in culture grade water with enough aliquots of the Cas 9 and sgRNA to allow the lab to make at least 3 mixes.
Guarantee: 1 founder
Fees: FVB injections: BIDMC investigator = $3,500
Non-BIDMC investigator = $5,250
C57Bl/6 injections: BIDMC investigator = $5,000
Non-BIDMC investigator = $7,500
The Basic Services of the Beth Israel Deaconess Transgenic Facility are:
- DNA Injections
- Embryonic Stem Cell Injections
- Embryo Freezing
- Line Rescue
In addition to the services the Transgenic Facility provides, the following techniques are available to the BIDMC/HMS research community:
- Embryo Culture
- Embryo Transfer
- Embryo Micromanipulation
- Mouse In Vitro Fertilization
- Mouse Artificial Insemination
- Mouse Vasectomies
- Microtool Development and Preparation
The first transgenic animal created in the facility was born in January of 1992. During that first year, ('91-'92) 15 constructs were injected. Other significant dates include:
Joel Lawitts, PhD, Director
Beth Israel Deaconess Transgenic Core
Instructor, Department of Pathology
Harvard Medical School