Generating CRISPR/Cas9-Derived Mutant Mice by Zygote Cytoplasmic Injection Using an Automatic Microinjector

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of producing materials in comparison to other methodologies (such as embryonic stem cells, ESCs) and the ability to simultaneously target multiple genes in one injection session. Here we describe a step by step protocol, from preparation of materials through to injection and validation of a cytoplasmic injection, which can be used to generate CRISPR mutants. This can be accomplished from start of injection to completion within 2-4 h with high survival and developmental rates of injected zygotes and offers significant advantages over pronuclear and other previously described methodologies for microinjection.

Keywords: CRISPR/Cas9; cytoplasmic injection; microinjection; zygotes.

Figure 1

Structures of the fertilized zygote. (a) zona pellucida; (b) oolemma; (c) cytoplasm; (d) pronuclei; (e) nucleoli; (f) perivitelline space; (g) polar bodies.

Figure 2

(a) Electronic micromanipulator for holding pipette; (b) electronic micromanipulator for injection needle; (c) inverted microscope; (d) Femtojet automatic injector; (e) instrument holder for injection needle; (f) instrument holder for holding pipette; (g) manual microinjector for providing positive and negative pressure to holding pipette.

Figure 3

(a) Hold the zygote and (b) ensure that the cytoplasm is held into the holding pipette (this ensures the embryo will not rotate during the injection process); (c) insert the injection pipette into the zygote avoiding the pronucleus and polar bodies and pause briefly halfway inside the egg to see the formation of a small droplet around the injection tip (arrow). This shows the CRISPR/Cas injection mix is flowing; (d) push the pipette forward again until it reaches the opposite side of the oolemma, pass the pipette tip through the oolemma to break the membrane and draw back into the embryo; (e) look for movement inside the cytoplasm to signify the CRISPR/Cas injection mix has successfully been injected inside the embryo (arrow); (f) rapidly withdraw the pipette tip; (g) if you need to widen the tip slightly due to clogging, glance the injection pipette tip on the side of the holding pipette to break slightly or inside the holding pipette opening. Adjust down the PC to 15–25 hPa and increase as necessary; (h) showing embryos that have lysed post-injection with cytoplasmic content leaking into the perivitelline space.

Figure 4

Microinjection dish set up. Dish showing a depression slide filled with flushing holding medium (FHM). Uninjected embryos in the centre of the dish. Injected embryos are moved to the top of the dish. These should be evaluated after injection and lysed embryos removed.