CRISPR-Cas9 has become a magic tool for molecular biologists, transforming genetic engineering from a once unbelievable dream into a tangible reality. Today, you can easily edit primary cells or cell lines within a few weeks with well-established protocols or others’ hands-on advice.
However, no matter how many success stories you find online or hear at seminars or conferences, it is often painstaking to find an effective and reproducible protocol for CRISPR-Cas9 editing in Drosophila melanogaster. Unfortunately, the well-established protocols are often not applicable to fruit flies.
In this article, I’d like to share with you an efficient strategy, originally developed by Port et al.,3 that we adopted to successfully make three different CRISPR-Cas9 knock-in fruit flies.
Challenges in Performing CRISPR Cas9 in Drosophila
- You need an effective way to get your custom-designed gRNA and Cas9 into fly embryos to induce a double-strand break at the target genomic locus.
- If your goal is to knock-in a gene, you will also need to inject a donor DNA repair template. In this case, you will need to inject three components (gRNA, Cas9, and donor DNA) as separate plasmids or RNAs into embryos. Because these elements will be only transiently expressed after injection, a lengthy optimization process for ratios, concentration, and forms (DNA or RNA) of gRNA and Cas9 is inevitable.
- Even after you have successfully optimized your protocol, variation in editing efficiency will likely occur from time to time. This can be due to inconsistent RNA quality, the actual amount of reagent injected, and the health of the embryos. Therefore, it is plausible that your success rate might increase if you eliminate the need to inject gRNA and Cas9, and instead inject only the donor DNA vector.
A Game Changer — the Transgenic CRISPR/Cas9 Strategy (a Fly Expressing Cas9 and gRNA)
The fruit fly, sometimes referred to as the golden bug, is a very attractive model to study the function of any gene of interest in a temporal and tissue-specific manner, because of the ease of introducing and removing genes from the fly genome. Therefore, it is hardly surprising that fly researchers are striving to incorporate CRISPR into their molecular biology toolbox.
To circumvent the challenges outlined above, research groups throughout the world have generated a number of transgenic Cas9 flies with stable tissue-specific Cas9 expression. Crossing a fly that expresses a customized gRNA with a Cas9-expressing fly should yield embryos expressing the gRNA and Cas9 with improved CRISPR activity.3 Once you reach this point, you only need to find a way to inject donor repair template DNA into the embryos. This approach simplifies your work and proves to be more efficient and reproducible than injecting gRNA and Cas9 as separate plasmids along with the donor DNA.3
Which Cas9 Fly Should You Use?
The transgenic CRISPR/Cas9 strategy is efficient and consistent. However, the improved CRISPR activity may result in increased mortality. If the genes that you want to edit or target your reporter to are essential in the fly, you are very likely to observe lethality once you cross your gRNA fly to a fly that ubiquitously expresses Cas9.
In this case, it would be wise to restrict the CRISPR activity to the germline so that the edited genotype can be passed down to the progeny, and so that you can prevent damaging the genomes of somatic cells. In my experience, nos-Cas9 (Cas9 driven by the germ-line specific nanos promoter) strains offer decent and germline-specific Cas9 activity with only modest effects on fly survival.
What Is the Ideal Configuration for Donor DNA?
When trying to decide upon the ideal configuration for donor DNA, you will need to consider the following:
- What is the ideal size of the homology arms?
- What is the better form (circular plasmids, linearized plasmids, PCR products, or synthetic single-stranded oligonucleotides)?
To address these questions, Dana Carroll and colleagues at the University of Utah systematically analyzed the required length of homology arms and different configurations of donor DNA for effective homologous recombination in fly embryos.1,2 They concluded the following:
- A minimum length of 500 bp homology in a circular plasmid is required for successful homologous recombination. With this, 2% of injected flies will have the desired mutation.
- 1,000 bp can provide additional efficiency, with 8% of injected flies mutated correctly.
- Maximal efficiency of 20% mutation observed with 2,000 bp or longer versions.
- A circular donor DNA is more effective in generating knock-ins than a linear template or a PCR product (2)
Bear in mind, however, that these parameters also depend on the size of your insert. If your goal is to introduce a small piece of DNA, such as a HIS-tag, attP site, or a subcellular localization signal, a donor DNA with short homology arms might be sufficient. On the other hand, if you want to incorporate a larger gene (e.g., GFP), you should consider using a long homology arm version.
Donor DNA – My Experience
As part of the daily work in our group, we frequently insert an 800 bp fluorescent reporter such as GFP into different target loci in the two widely used Drosophila cell lines, S2 and Kc. In cell lines, we often see comparable integration efficiency with 700 and 1,400 bp homology arms in a 3.3kb backbone circular donor DNA.
However, when it comes to making a real GFP knock-in fly, we have thus far only tried the 1,400 bp homology version and it resulted in a high success rate (with 30–50% of the offspring carrying the knock-in). Donor DNA in the form of PCR product failed to generate any successful knock-in progeny.
Which Template Should You Use to Make Your gRNA?
Different promoters drive gRNA expression with different efficiencies. The U6:3 promoter was shown to be overwhelmingly better than U6:2 in inducing transmissible mutation.3 In our hands, there was no obvious difference in activity between these two promoters in S2 or Kc cell lines. We chose the U6:3 promoter gRNA to generate our knock-in flies. Both of these plasmids can be requested from Addgene (Plasmids #49410 and #49409).
Outsourcing the Injection?
The creation of gRNA flies requires a standard attB–attP site-specific integration. This is a standard procedure that most injection companies offer with high success rates at a low cost. Unless you already have the appropriate setup and well-trained personnel, outsourcing the injection to professionals is a cost-effective option.
You can test your gRNA efficiency in S2 or Kc cell lines before making the transgenic gRNA fly you want. Although this can vary depending on the loci and insert size, having our gRNA tested and verified in these cell lines gave us good success rate in creating knock-in flies (with 30–50% of progeny carrying the gene of insert).
Do you have any additional tips for CRISPR in fruit flies, or indeed other insects? Please share your experiences with us by writing in the comments section!
- Beumer, KJK, Trautman JKJ, Mukherjee K, Carroll, D. (2013). Donor DNA Utilization During Gene Targeting with Zinc-Finger Nucleases. G3: Genes| Genomes|Genetics 3:657–64.
- Beumer KJ, Trautman JK, Bozas A, Liu JL, Rutter J, Gall JG, Carroll D. (2008). Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci U S A 105(50): 19821–6.
- Port F, Chen HM, Lee T, Bullock SL. (2014). Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A 111(29): E2967–76.