With such a well-studied and tractable model organism as C. elegans, there are many choices when it comes to the method you use to identify your transgenic animals. Choosing the best phenotypic screening method for your transgenic project is critical, as it can potentially impact the phenotype of your final transgenic strain.
In this blog, we will discuss key factors to consider when choosing a screening method, introduce various editing techniques, and discuss the pros & cons of various screening methods.
There are several things to consider when choosing a screening method, including:
- Which genome editing method you’re using
- which tissue your gene of interest is expressed in
- how the phenotypic screening method could potentially interact with your transgenic edit.
Different genome editing techniques are more compatible with some phenotypic screening methods than others. When editing the genome with CRISPR/Cas9, there are several options for your screening method. One option is to use a co-CRISPR edit. This is an edit of a locus that generates a known phenotype, separate from your target edit. This option allows you to easily visually identify animals in which the CRISPR/Cas9 system was active. These animals are up to 10 times more likely to contain your desired edit (Kim et al., 2014). Another option is to use a selectable marker that is incorporated into your target locus, along with your desired edit. While this method is helpful for edits that may be difficult to identify and/or isolate, it does not indicate whether the CRISPR/Cas9 system was active in the animals displaying the phenotype conferred by the selectable marker. For example, if the animals with the target edit are not viable as homozygotes, it is far easier to maintain a heterozygous population when the selectable marker is integrated at the target edit locus.
One great benefit of using a co-CRISPR edit to identify your transgenic animals is that the co-CRISPR edit will not be in your final strain. The only modification to the genome of your final strain will be your target edit. If using a co-CRISPR edit is most appropriate for your transgenic project, there are a few edits to choose from, resulting in various phenotypes. The edit to the dpy-10 gene, described by Arribere et. al, is a popular and useful co-CRISPR phenotypic screen. This method edits the dpy-10 gene to create rolling animals when the edit is heterozygous or dumpy animals when the edit is homozygous. This screen is easily visually identified and works quite well. Another co-CRISPR option is the unc-18 edit. In this case, you begin with a C. elegans strain that already has an unc-18 null edit. The co-CRISPR edit in this case rescues the unc-18 null edit, returning it to wildtype. This is helpful in several ways. First, there is no co-CRISPR phenotype that you need to screen away from in your final strain, which can save time. Second, there is an easy PCR screen you can use to determine that your strain is repaired to wildtype at the co-CRISPR locus. In contrast, in order to genotype the dpy-10 locus, sequencing is the best option, which takes longer and is more expensive than PCR.
InVivo Biosystems has a kit containing the unc-18 co-CRISPR reagents and injection strain for those who are interested.
As mentioned above, an alternative to using a co-CRISPR edit in your CRISPR/Cas9 project is to use a selectable marker that is integrated into the genome along with your target edit. This method of using a selectable marker is also employed in the MosSCI genome editing technique. The selection marker used in MosSCI edits is unc-119 rescue. Unc-119 rescue can be used as a selectable marker whether you are editing the genome with CRISPR/Cas9 or with MosSCI methods. The unc-119 rescue selectable marker rescues a null unc-119 C. elegans strain with a functional copy of the unc-119 gene. This phenotypic screen is very easy to use, as animals that have the selectable marker in their genome return to crawling from being uncoordinated. One drawback of the unc-119 selectable marker is that it is neuronally expressed (Maduro et. al, 1995). If the gene of your target edit is also neuronally expressed, then the ectopic expression of the unc-119 gene could be problematic when you are studying your final strain. For this reason, InVivo Biosystems always uses a different selection marker when the gene of interest is neuronally expressed.
In the case that unc-119 rescue is not ideal for your project, there are several other selectable markers to choose from. At InVivo Biosystems, we frequently choose hygromycin resistance as our selectable marker. This selectable marker can be a bit leaky until you optimize your protocol. Once the protocol is optimized, hygromycin resistance is a fairly easy to use selectable marker. There are additional antibiotic resistance selectable markers such as neomycin (Giordano-Santini et. al, 2010), pha-1, cha-1, and lin-15. While we don’t regularly work with these selectable markers, we have worked with a number of clients who prefer to use them in the past and are open to working with you with whichever marker you prefer.
Co-Injection/Extrachromosomal Array Markers
Co-injection markers, or array markers, are key to finding extrachromosomal array lines. They are also helpful in the case that you don’t have the appropriate co-CRISPR reagents available, however, you might have to screen more animals. Co-injection markers let you know your injection was good in a particular set of animals. This can help to narrow your search for your transgenic animals. Some of the markers that have already been mentioned are suitable for use as co-injection markers, such as unc-119 rescue and hygromycin resistance. Rol-6 is another great co-injection marker. The rol-6 plasmid produces a roller phenotype when expressed (Mello et al., 1991). We have an optimized rol-6 that is available as a plasmid. Fluorescent markers are another great co-injection marker to use because they are easy to identify. We have a handful of fluorescent markers that we like to use regularly, however, we are able to make custom fluorescent markers as needed for client projects. One thing to note is that if you are using a fluorophore in your project, it’s recommended that you use a different fluorophore as your fluorescent array marker in order to easily distinguish between the two fluorescence patterns.
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