Efficient production of CRISPR/Cas9 gene knockouts in the male/female nematode Caenorhabditis nigoni

Abstract

Although nematode genetics was founded on the use of hermaphrodite genetics for studying animal development and behavior, there is a growing need to extend this work to male/female species. One of the most promising species is C. nigoni, because it is so closely related to the model hermaphroditic C. briggsae. We present methods for using CRISPR/Cas9 gene editing to create mutations, and techniques for balancing, maintaining and studying these mutations.

Figure 1. Workflow to produce and identify Cas9-induced gene knockouts in C. nigoni

This workflow represents the steps needed to produce directed genome edits in C. nigoni . (A) Select young gravid females for microinjection, to ensure large and reliable F ₁ broods. Ideally, there should only be a few embryos in the uterus and a visible mating plug (previously deposited by a male over the vulva). (B) Use standard techniques for microinjection of Caenorhabditis nematodes (Berkowitz et al. , 2008). The injection mixture should contain Cas9/sgRNA RNP complexes (Paix et al. , 2014), a selection marker (pMyo-2::GFP plasmid) and a repair template, if one is needed. (C) If possible, co-inject an easily detectable selection marker. We use a GFP expression plasmid controlled by the pharyngeal myosin promotor, pMyo-2, and screen the F ₁ progeny with a fluorescence stereo microscope to identify P ₀ females that were successfully injected. (D) Cross pairs of F ₁ males and females to produce F ₂ descendants, then harvest the parents of successful broods for PCR, and screen them for indels. Use as many sibling pairs as possible. Cross surplus F ₁ animals with wildtype animals of the opposite sex. After reproduction, lyse the F ₁ parents to isolate genomic DNA. Amplify DNA from the target site (~100 bp) using standard PCR methods and screen for a mobility shift on 8% acrylamide gels. This approach can be used to detect small insertions or deletions, or precise edits such as a tag or new restriction site. Roughly 2/3 of the indel mutations result in a frameshift, which should produce a premature stop codon. Sequence mutant alleles promptly to avoid expending resources on undesirable mutations. (E) Following identification of a new mutant, use sibling crosses to make homozygotes. Alternatively, use crosses with animals that carry a balancer mutation to establish a stable heterozygous strain. (F) Once homozygotes are identified they can be studied for their unique properties, like this C. nigoni dpy-18( v484 ) mutant.