CRISPR/Cas9 mutagenesis in Volvox

Researchers in Stephen Miller’s lab at the University of Maryland, Baltimore County have successfully used CRISPR/Cas9 to knock out several developmentally important genes in Volvox carteri. CRISPR/Cas9 is a relatively new technology that allows heritable mutations to be introduced into living cells at specific locations within the genome.

This advance was announced in a new paper in The Plant Journal by José A. Ortega-Escalante, Robyn Jasper, and Stephen M. Miller (Jasper and Ortega-Escalante are listed as equal contributors). They were able to transform wild-type V. carteri with inversion-deficient and somatic-regenerator mutations, and they transformed somatic regenerator mutants with a gonidialess (no specialized reproductive cells) mutation.

I have never used CRISPR/Cas9, and I don’t know as much about it as I should, so I’m sure any explanation I gave would be riddled with errors. Here’s someone who seems to know what she’s talking about:

That’s Jennifer Doudna, co-inventor, with Emmanuelle Charpentier and Virginijus Šikšnys, of the CRISPR/Cas9 system. For their roles in CRISPR/Cas9 development, Doudna and Charpentier have shared the 2015 Breakthrough Prize, the 2015 Gruber Prize in genetics, the 2016 Warren Alpert Prize (with Šikšnys and two others), and the 2018 Kavli Prize in Nanoscience (with Šikšnys).

Briefly, CRISPR/Cas9 is a bacterial mechanism for destroying viral DNA, and humans have exploited it into a system for making specific changes at precise locations within the genomes of living organisms. In the video above, Dr. Doudna is focused on repairing malfunctioning genes, but CRISPR/Cas9 is equally effective at introducing malfunctions to (previously) normally functioning genes, which is often a useful tool in basic research. The reason we often want to do this is that by breaking a gene and observing the result, we can often get an idea of the gene’s function.

In a sense, Ortega-Escalante, Jasper, and Miller turned this logic on its head. Rather than using CRISPR/Cas9 to identify gene functions, they used known gene functions to assay the success of their transformations. By targeting three genes with known, easily observable phenotypes, they were able to identify Volvox spheroids in which CRISPR/Cas9 had introduced loss of function mutations:

Ortega-Escalante, Jasper, Miller 2018 Fig 3B-D

Figure 3B-D from Ortega-Escalante, Jasper, and Miller 2018. B: somatic regenerator mutant (left) and gonidialess transformant of somatic regenerator mutant (right), C: wild-type adult V. carteri (left) and somatic regenerator transformant (right), D: wild-type juvenile V. carteri (left) and inversionless transformant (right). Scale bar 100 μm.

I was going to say “they were able to easily identify,” but I don’t think it was easy. For the gonidialess experiment, they observed ~100,000 spheroids for each of fourteen lines:

Spheroids from seven G4 and seven G7 transformant lines were cultured in UFSVM flasks to induce Cas9 expression. After 6 days of induction (2 generations), all the G7 transformant progeny appeared to develop normally with respect to gonidial number, but three of the G4 transformants each gave rise to 5-10 Gls progeny, out of ~100,000 examined for each line.

This low efficiency of mutagenesis is problematic for the kind of reverse genetics applications I mentioned earlier (“breaking a gene and observing the result”). Most mutant phenotypes will not be as dramatic as the gonidialess, somatic regenerator, and inversionless mutants in Figure 3 above, and the prospect of screening 100,000 progeny to find a handful of mutants with some subtle or unknown phenotype is daunting. I asked Dr. Miller why he thought they found so few mutant progeny, and he kindly allowed me to quote his answer:

Others working with CRISPR in other systems have reported similarly variable rates of mutagenesis. I think the most likely reasons for variability are that 1) different guide RNAs have different efficiencies, as there appear to be preferences for certain bases at each of the 20 different positions in the guide RNA sequence, and 2) chromatin structure plays an important role in determining whether or not Cas9 gains access to the target region. We can make some educated guesses about the former with respect to guide RNA sequences that will work best, based on studies others have done in other model systems, but chromatin structure is impossible to predict (though I believe some people are using ChIP-seq data to predict which regions of a gene are likely to be more accessible than others to the Cas9 nuclease).

The authors suggest a strategy to mitigate this limitation:

Previous studies have revealed several genes responsible for some of the developmental complexity that has arisen in this family, and other studies have produced an impressive number of candidate genes that might also have played important roles (Duncan et al., 2007; Ferris et al., 2010; Prochnik et al., 2010; Arakaki et al., 2013; Klein et al., 2017; Matt and Umen, 2018). The complexity of traits under investigation by the volvocine algal research community include, but are not limited to, cell differentiation, cell division number modulation, embryo morphogenesis, cell wall/extracellular matrix architecture and deconstruction, and gametogenesis. It should now be possible to do rapid functional analysis of genes that are strong candidates to contribute to these traits based on their expression patterns or homology to known regulators of these traits.

This makes sense to me. In cases in which a candidate gene has been identified through other methods and there is a suspected phenotype, their protocol should allow identification of mutant progeny, at least when the mutants are easily distinguished (visually or otherwise). The authors acknowledge that this won’t always be the case, though:

…it is likely that in many/most cases the mutant phenotype associated with a targeted gene will be unknown, making it challenging and time consuming to find and test putative mutants when they appear with low frequency, as they did for glsA and invA. Therefore, it would be advantageous to improve the current system to make mutagenesis and identification of Cas9-generated mutants more efficient.

They suggest several possible improvements to overcome this limitation and broaden the utility of their protocol. One suggestion that’s particularly exciting to me is that this technology might be straightforward to apply to other volvocine species, for which we know next to nothing about their developmental genetics:

It should also be possible to modify this Cas9 system to interrogate gene function in other species of Volvox and/or colonial volvocine algae, to gain a better understanding of how and when gene functions may have evolved in this family.

This is potentially a big step forward in Volvox genetics. All the cool kid model organisms have CRISPR/Cas9: yeast, chicken, mouse, C. elegansArabidopsisChlamydomonas, etc., and it’s a powerful technology allowing unprecedented control of genome editing. I look forward to seeing the implementation of some of the improvements suggested by Ortega-Escalante, Jasper, and Miller, and I’m optimistic that their protocol will accelerate progress in understanding the developmental genetics, and ultimately the evolution, of the volvocine algae.

Stephen Miller is an Associate Professor in Biological Sciences at the University of Maryland, Baltimore County. Both of the lead authors were students in the Miller lab when they were working on this project. Robyn Jasper, who was an undergraduate during this work, is now a PhD student in Plant and Microbial Biology at the University of California, Berkeley. José Ortega-Escalante, who was a graduate student, successfully defended his Ph.D. thesis the very next day after this paper was published online, and he has moved on to a job with the Maryland Department of Public Health.


Stable links:

Ortega-Escalante, J. A., R. Jasper, and S. M. Miller. 2018. CRISPR/Cas9 mutagenesis in Volvox carteri. The Plant Journal (online early), doi: 10.1111/tpj.14149.

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