The Importance of Crispr/cas9 and Methods and Dangers of Gene Drive

Abstract

This paper looks at the overall process of gene drive and its methods. First the reader will be given a brief overview of the concept of gene drive along with a description of how gene drive systems can be used to manipulate patterns of mendelian inheritance. The focus of this paper will be on the methods of gene drive, the importance of CRISPR/Cas9, and the dangers of gene drive.

Gene Drives

Gene drive is a method whereby geneticists seek to modify the normal patterns of mendelian inheritance within a population. Gene drive works on mendelian inheritance patterns via two different processes. In the first process, homing, a desired allele copies itself onto its homolog in place of the wild-type allele which leads to a higher number of offspring with that allele (Champer, Buchman & Akbari., 2016).

The second process works by lowering the viability of gametes containing wild-type alleles versus gene drive alleles, thereby lowering the frequency of wild-type alleles in the population. The aim of gene drives, in general, is to either push a desired trait though a population (Modification) or to suppress/eliminate a population (Suppression) (Champer et al., 2016). Gene drives work to achieve desired results through a number of different methodologies.

Proposed methods of Gene Drive

One proposed method for gene drive seeks to mimic a naturally occurring process whereby specialized genes seek out and target a gene on the opposing chromosome. The genes, homing endonuclease genes (HEGs), code for a protein that binds to a specific sequence of nucleotides and cleaves DNA at that site. Gene drive systems based on this process are collectively known as homing-based drives (Champer et al., 2016).

HEGs that occur naturally do not allow for targeting of specific genes. The task of identifying all possible naturally occurring HEGs and tailoring them to the individual needs of geneticists would be monumental. Instead, geneticists have been looking to new technology that allows the creation of recombinant HEGs that can be made to target any desired gene within the genome of a species (Champer, et al., 2016). This proposed method, CRISPR/Cas9, will be discussed later in this paper.

Homing-based drives work by forcing the repair of DNA via natural processes. The strand may repair without the aid of a template strand by ligating the broken ends of the strand back together, this pathway is known as non-homologous end joining or NHEJ (Gilles & Averof, 2015). If NHEJ occurs the target gene will simply be excised and the strand will be repaired with the gene removed. Repair may also occur with through the use of the template strand via homology directed repair or HDR. In HDR the HEG will serve as a template for the repair of the targeted strand, this will result in the HEG present in both of the homologous chromosomes (Champer et al., 2016).

In either event the normal patterns of mendelian inheritance will have been altered, due to the fact that homing-based drives work during meiotic cell division (Champer et al., 2016). If NHEJ occurs, the target DNA will have reduced viability compared to the HEG containing DNA. Overall this leads to a lower frequency of the wild-type allele which means the HEG functions as a suppression gene drive. HDR has the added benefit of spreading the mutant allele and reducing expression of the wild-type allele. Homing-based drives are desirable as a method of gene drive due to this unique nature. Of the proposed methods of gene drive, homing-based drive is the only method capable of both suppression and modification of a population (Champer et al., 2016).

Another proposed method of gene drive, sex-linked meiotic drive, aims to suppress a population by altering the number of male to female progeny. Sex-linked meiotic drives are similar to homing-based drives as they contain an endonuclease carrying gene. This gene is sex-linked to males of a species. In the presence of an X-chromosome during meiotic division the endonuclease produced by the gene targets and cleaves the X-chromosome at multiple locations. Any X-chromosome containing sperm will then be nonviable (Champer et al., 2016). Genes that act in this manner are known as X-shredder genes (Champer et al., 2016).

Any progeny of males that carry X-shredder genes will also be male. If the X-shredder genes is located on autosomal chromosomes then there is a chance that the offspring will inherit X-shredder. If instead the X-shredder gene is carried on the Y-chromosome then all offspring of that male will carry the gene (Champer et al., 2016). Populations in which X-shredder alleles spread will see a stark reduction in the number of female progeny. Eventually the population will not be able to sustain itself, resulting in a successful suppression gene drive. Unfortunately X-shredder genes do not exist for all species, geneticists hope to create recombinant X-shredder genes using the same method proposed for homing-based drives.

The last proposed gene drive method discussed in this paper, is maternal dominant embryonic arrest (Medea). Medea occurs in females of a species during oogenesis (Champer et al., 2016). It was first identified in beetles. The mother insect carries a gene that expresses a toxin during oogenesis. Offspring that inherit the gene also inherit a gene that produces an antidote in the early zygotic stage of development. Zygotes that do not carry the gene for Medea are unable to produce an antidote and die during development (Champer et al., 2016).

Medea carrying females select for Medea carrying offspring. Proposed gene drives with Medea aim to exploit this process with recombinant DNA. By inserting the desired gene and creating a unique toxin and antidote combination using CRISPR/Cas9 technology geneticists will be able to quickly spread a gene through a population.

Of all the methods listed, natural processes created the framework that geneticists are seeking to exploit. A logical improvement to any gene drive is the ability to target any gene as well as insert and desired gene into the genome of the target organism. The emergence of CRISPR/Cas9 technology promises to do just that.

CRISPR/Cas9 and gene drives

Clustered regularly-interspaced short palindromic repeats (CRISPR) along with CRISPR associated protein 9 (Cas 9) is an endonuclease system that allows geneticists to target any desired gene through the use of specialized guide RNAs (Gilles & Averof, 2015). An endonuclease is a protein that cleaves DNA. CRISPR is a system that bacteria use to combat phages. Bacteria are able to integrate viral DNA into it’s own genome, that viral DNA is then expressed in the form or RNA and is coupled with an endonuclease (Wade, 2015). A team of researchers were able to modify CRISPR to allow for targeting of any sequence of DNA.

CRISPR/Cas9 uses custom made guide RNAs (gRNAs) that can target any sequence they desire. The appeal of CRISPR is it’s ease of use, low cost, and potential applications (Gilles & Averof, 2015). Specifically, in regard to gene drives, it allows for the customized targeting of unwanted genes. Also by utilizing the entire CRISPR/Cas9 complex geneticists have the option of introducing any gene into a host’s genome. Homing-based drives could be tailor-made to target very specific genes. CRISPR/Cas9 allows for specificity to a sequence as short as 20 nucleotides in length (Gilles & Averof, 2015).

The proposed drive method would then do the work of propagating that gene through the population in one generation. X-shredder and Medea recombinant genes would work in the same manner, just with added specificity. The work of trying to isolate unique X-shredder and Medea genes would no longer be an issue. With CRISPR/Cas9, X-shredder or Madea genes could potentially be introduced to any species. Creating the parental generation that would be used to begin the gene drive. Unchecked gene drives could prove to be problematic. As with any modification of genomes the potential dangers of gene drives have not gone unnoticed.

Dangers and precautionary measures

The greatest risk posed by gene drives is the potential damage a drive could have on an ecosystem. Gene drives have the ability to proliferate through a population in a little as one or two generations (Wade, 2015). The danger of an off-target effect can be extremely profound. There is an inherent danger of mistakenly crashing a population by releasing a gene drive carrying organism in an area that cannot handle it (Wade, 2015).

Another concern lies with the potential for unintended consequences. Without careful consideration of what the impact of significantly reducing a population may do, biologists risk dealing with ripple effects. Eliminating a keystone species from an area could snowball into a larger issue with animals that are higher up on the food chain (Wade, 2015). Another possible impact of gene drives comes from the potential mutation and passing on of that gene to a sister species or mutated pathogenicity (Wade, 2015).

To combat the potential for disaster, biologists have thought of ways to end a rogue gene drive. Gene drives are classified based on the ability to remove or reverse that drive (Champer et al., 2016). A “standby reversal drive” is a cautionary measure that biologists can take when designing gene drives. A reversal drive is a secondary gene drive that has the ability to stop the spread of the primary gene drive (Champer et al., 2016). Another precaution has been the establishment of guidelines pertaining to gene drives by the the US National Academy of Sciences (Champer et al., 2016).

Conclusion

The main focus of gene drives has been the potential to use gene drive as a mechanism to eliminate disease and pestilence. One of the most promising application of gene drive has been in the attempt to use gene drive to eliminate malaria in mosquitoes. In 2015, researchers from the University of California showed that they were able to successfully introduce anti-malaria genes into a species of mosquito using Cas9 (Gantz et al., 2015).

A big hurdle for gene drive currently is the development of successful protocols. Although the group from the University of California showed it was possible to use Cas9 to create anti-malarial gene drives, they did so with much difficulty. As it turns out Cas9 is toxic to mosquitoes, so the group had to develop novel ways of mitigating that toxin to successfully insert the gene (Saey, 2015). Another noted issue was the propensity for off-target cuts while using Cas9 and certain gRNAs (Saey, 2015). The reality of gene drives is only just beginning, CRISPR/Cas9 has taken gene drive from a simple concept to a coherent process.