CRISPR for Conservation

I’ve got this feeling that CRISPR is the next PCR.  Have you ever met someone who was an early adopter of PCR?  No, I mean an early adopter of PCR where the technique required three water baths, a swivel chair, a stop watch, and AN ACTUAL PERSON to move the reaction tubes between water baths every 30 seconds.  Now it’s so common PCR is undergrad grunt work.  That’s how I feel about CRISPR, like it will be undergrad grunt work in 20 years, so I better stop ignoring and start incorporating it into my science.

What is CRISPR?
Clustered Regularly Interspaced Short Palindromic Repeat

CRISPR is a natural immune response in bacteria to defend against viruses.  It cuts both strands of the DNA of an invading pathogen, thereby disrupting replication and gene expression.  Molecular biologists took this idea and turned it into a tool for gene editing.  And not blunt force trauma gene editing, precision editing: removing or adding a gene, editing a single base pair, increasing or decreasing the expression of a gene, and editing methylation and phosphorylation sites.  This is powerful technology that could elucidate both gene function and how different alleles of the same gene produce variation (aka- phenotypes).  The precision could also allow us to study how genes interact with each other.

CRISPR is really a single strand of RNA that contains several base pairs of sequence identical to the virus DNA.  The CRISPR sequence associates with a protein called Cas9.  When the CRISPR-Cas9 complex finds a virus, Cas9 will un-twist the virus DNA, then the identical bases on the CRISPR strand can anneal to the virus, once this happens Cas9 will cut the virus DNA.  Like our immune systems, a bacteria can have many different CRISPRs to identify unique viruses.  Molecular biologists realized they could manipulate this system to silence genes, then adapted it to cut DNA and insert new sequences making it a precision genome editor.

A schematic of the CRISPR-Cas9 system. The CRISPR RNA molecule (purple) matches a region of genomic DNA of the target organism (pink) and associates with it in the Cas9 protein. Once the complex has assembled, Cas9 cuts the genomic DNA. The cut DNA should repair itself incorrectly, thus deactivating the gene; however, variations on construct design can insert a target sequence thus editing the target DNA sequence.
Source: Australian Broadcasting Corporation

Scientists are very excited about how CRISPR will allow us to understand the function of genes.  While gene knockouts, knock-downs, and over-expression studies have been around for decades, they are time and money intensive experiments to set up.  CRISPR offers a targeted and cheap way to alter a gene’s sequence or expression then observe the effect on phenotypes.

How can conservation use CRISPR to support biodiversity goals?
The first challenge is to get a CRISPR construct into an individual.  Two approaches here, first is to directly insert the construct into an embryo early enough during development that all resultant cells would contain CRISPR.  The second approach is to do the first approach, then let those individuals breed and pass on the CRISPR architecture within a population.  Both approaches suffer from the problem that very few individuals within a population would get the benefit conferred from CRISPR.  But, if the CRISPR construct is linked to a gene drive, then the inheritance by future generations will speed up.  Gene drives take advantage of “selfish genes” which promote their own inheritance, thus have a higher probability of inheritance than Mendelian traits (50% chance for either allele from one parent).

Controlling/eradicating the spread of disease is the most commonly discussed use of CRISPR in nature.  Most current work on this front is in the mosquito-malaria system, where mosquitoes are made to be resistant to carrying malaria.  These resistant mosquitoes would be released into the wild and over time the mosquito population would carry less malaria thereby decreasing transmission to humans.  This idea extends to pathogens attacking threatened species such as the fungal chestnut blight, chytrid ravaging diverse species of amphibians, or Sylvatic plague in black-footed ferrets.  If we can target CRISPR to disrupt pathogen genes important to their replication, transmission, or virulence, there may be a mechanism to decrease pathogen load to protect threatened and endangered species.

Controlling/eradicating invasive species is another potential application.  CRISPR could be used to disrupt reproduction of invasive species.  One way would be to disrupt genes important to either sperm or egg formation thereby decreasing the number of gametes in a population.  Similarly, disrupting genes active in early development may result in aborted embryos.  However, a different approach would be to skew the sex-ratio of a population to have more males; with fewer females in the population each generation, the birth rate would decrease over time.  All of these strategies are meant to force a population crash, but they rely on disrupting reproduction over multiple generations.  This could be a very long game for some invasive species; take pythons in the Everglades which may not be reproductively mature until ~6 years old then have lifespans 20-25 years.  As gene drives may take dozens of generations to spread through a population, using this approach on long-lived species may require centuries before a crash.  And this doesn’t even account for new migrants into the system, such as released pet snakes in the Everglades.

Increasing genetic diversity has also been proposed as a way for CRISPR to contribute to threatened and endangered species conservation.  Many endangered species have low genetic diversity meaning they may not be able to adapt to changing conditions such as new pathogens or climate.  Increased genetic diversity could be targeted to specific genes, perhaps those related to immune response, for which scientist suspect will help a population adapt.  This is a particularly interesting idea when thinking about how adding in alleles from past populations, identified using ancient DNA, could increase diversity known to be absent from contemporary populations.  Alternatively, CRISPR has been proposed as a mechanism to generate random mutations in the genome which natural selection may act upon in the future.  Similarly, deleterious alleles could be edited particularly when they code for non-conservative amino acid changes or premature stop codons, thus dramatically altering gene folding or length.  Editing deleterious alleles could increase survival in some endangered populations

Kink in the tail of a Florida panther, due to inbreeding. Panthers have very low fitness and multiple traits including those for reproduction are affected. Could CRISPR increase genetic diversity and remove deleterious alleles (such as the one producing the kinked tail phenotype)?
Source: University of Florida Extension

I’m ignoring much of the complexity of the issue to focus on the thought experiment about how CRISPR could enhance the conservation of biodiversity (for more reading see this paper and references within). Part of that complexity is evolution itself. Given that the approaches mentioned are multi-generation, selection could act to decrease the efficacy of the gene drive, or biological systems could compensate in unique ways (i.e. parthenogenesis, hybridization, or increased virulence- life always looks for a way). This further ignores ethical issues as well as public acceptance of this type of genetically modified organism (GMO).

Side photo of a Florida panther (Puma concolor coryi) by Lynn Stone via ARKive.

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