10 essential considerations for creating mouse models with CRISPR/Cas9

The Jackson Laboratory

CRISPR/Cas9 technology applied to generating mouse models is a very recent revolutionary method. Here we discuss 10 basic stages needed to create mouse models using this technology.

Wouldn’t it be nice to create mouse models without the struggle and the agonizing wait?

At JAX we are fortunate to have Dr. Haoyi Wang on board bringing his remarkable expertise using the CRISPR/Cas9 system to engineer mutant mice.  As a post-doc in Dr. Rudolf Jaenisch’s group at the Whitehead Institute, Dr. Wang and his colleagues were the first scientists to generate mice with multiple genes knocked out using a one-step approach. He was able to generate these and other amazing models in a matter of a few weeks, instead of in a couple of years using traditional methods.

Why is this technique relatively fast and simple?

I will spare you the details of how bacteria and archaea use their endogenous CRISPR/Cas system to protect themselves against invading DNA vectors, by chopping and molecularly “remembering” the foreign DNA.

What is important is that Dr. Wang demonstrated that components of the CRISPR/Cas9 system can be microinjected directly into the embryos to get targeted gene editing with a very high efficiency.  This simple technique eliminates the necessity to engineer targeted mutations in embryonic stem cells, and the long process of creating chimeric mice from them by blastocyst injection.

What do you need to get started gene editing using CRISPR/Cas9?

Below are 10 essential considerations for creating mutant mice with the CRISPR/Cas9 system:

  1. Design a site-specific RNA guide with minimal mismatch off target activity. There are several freely available software tools to design and evaluate target sites for use with CRISPR/Cas9, for example: CRISPR design and analysis from MIT or E-CRISP from the German Cancer Research Center.
  2. Synthesize and purify both capped Cas9 mRNA for the DNA endonuclease activity and guide RNA/s for specificity.
  3. Purify donor DNA oligo if you are planning to do a knock-in model.
  4. Generate viable one-cell embryos of the desired genetic background via superovulation followed by in vitro fertilization.  (Note:  not all strain backgrounds respond well to superovulation).
  5. Microinject mRNAs (and donor DNA if applicable) into the embryo.
  6. Culture the embryos until they reach the blastocyst stage (3.5 days).
  7. Induce pseudopregnant dams.
  8. Transfer the (injected) cultured embryos into a pseudopregnant dam.
  9. Once the pups are born, genotype tail snips from founder mice to confirm CRISPR/Cas9-mediated genomic modifications.
  10. Breed positive founder mice to confirm germline transmission of the targeted mutation.

Easier said than done, right?

The beauty of the CRISPR/Cas9 system, too, is that you can co-inject multiple guide RNAs to simultaneously create double or triple KO models. Traditional targeted mutation methods typically allowed for creating mutations one at a time.  Producing triple knockout mice, then, usually required interbreeding mice with the single knockouts – a labor-intensive effort that often required a year or more to produce triple homozygous mutants for study.

As we speak, many labs including Dr. Wang’s at The Jackson Laboratory are tweaking their protocols to find the optimal conditions to create complex models with precise gene disruptions with an even higher efficiency.

We’ll make sure to keep you posted.