CRISPR-based gene drive systems can bias inheritance of desired traits by cutting a wild-type allele and copying the drive system in its place. This system are able to spread genes particularly rapidly. Components of the CRISPR system can be tailored to replace alternative copies of a particular gene, ensuring that only the desired version is passed on to offspring. For example, a gene that prevents mosquitoes from carrying or transmitting the malaria parasite could be introduced to a very large wild population to reduce the incidence of the disease among humans. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles. And, using such CRISPR gene drives for genetic modification of entire species has ignited an intense debate about potential applications as well as risks of such approaches.
CRISPR-based gene drive (GD) systems function by converting drive-heterozygotes into homozygotes in the late germline or early embryo. First, a CRISPR nuclease (e.g. Cas9) encoded in the drive construct cuts at the corresponding wild-type allele-its target prescribed by an independently expressed guide RNA (gRNA), which containing a 20 bp sequence and is complementary to the target site within the genome-producing a double-strand break (DSB). This break is then repaired either through homology-directed repair (HDR), producing a second copy of the gene drive construct, or through a nonhomologous repair pathway (non-homologous end joining, NHEJ, or microhomology-mediated end joining, MMEJ), which typically introduces a mutation at the target site.
Although HDR repair predominates, the competing repair pathway NHEJ can generate small insertions or deletions (indels) at the gRNA binding site and/or associated PAM (protospacer adjacent motif) sequence that will be resistant to subsequent cleavage by the encoded endonuclease. Thus, the allele converts from a wild-type to resistant allele if it undergoes repair by a pathway other than homology-directed repair. Moreover, drive-resistant alleles are expected to exist in wild populations simply due to standing genetic variation. Even though the probability of NHEJ appears to be low, the creation of resistance alleles through NHEJ, and the subsequent selection favouring their spread, provides one mechanism whereby a targeted population might rebound following an initial decline in abundance.
Fig 1. Molecular Mechanism of CRISPR GD. A. Typical construction and function of alteration-type CRISPR GD systems; B. Inheritance of a GD.
Rresistance against the drive is one of the key obstacles to a successful gene drive approach. Theoretical studies have shown that the formation of such resistance alleles can severely limit the ability of a drive allele to spread in large populations, especially when it confers a fitness cost. Several additional strategies have been suggested to lower resistance potential. For example, a drive construct could contain multiple gRNAs targeting different sites, in which case resistance may need to evolve independently at all sites in order to halt the drive. However, with the high rates of NHEJ-repair observed among current constructs, such an approach would still be unlikely to be effective alone and would probably need to be combined with other methods, such as a better promoter. Resistance allele formation could also be suppressed by utilizing a shRNA gene as part of the drive designed to suppress NHEJ in the germline and early embryo.
The CRISPR-Cas9 gene editing system could provide a mechanism for effective population control by gene drives. These advances have generated tremendous excitement among the agricultural, conservation and health science communities because they offer potential solutions to costly, long-standing problems such as the control or eradication of invasive species and the suppression of animal vectors of human disease. With the CRISPR gene drive mechanism, a genetic payload could be rapidly disseminated throughout an entire population. For example, a functioning gene drive could provide a highly efficient means for controlling vector-borne diseases such as malaria, which is notorious for rapidly acquiring drug resistance to every newly-deployed drug. Combined with a gene drive, scientists could provide a promising new approach to fight this devastating disease.
Because gene drives propagate by replacing other alleles that contain a cutting site and the corresponding homologies, their application is limited to sexually reproducing species. Due to the number of generations required for a gene drive to affect an entire population, the time to universality varies according to the reproductive cycle of each species. Hence this technology is of most use in fast-reproducing species. Some now view self-replicating gene drives as a much needed 'silver bullet' for conservation science and question not whether this technology is viable but whether it should be used, citing the risks associated with the dispersal or human-mediated transport of gene-drive carriers beyond the laboratory or the population targeted for management. Using such CRISPR gene drives for genetic modification of entire species has therefore ignited an intense debate about potential applications as well as risks of such approaches.
Genetically engineered animals normally come with few ecological risks. Most engineered traits are for human beneﬁt and will not be favored by natural selection. By contrast, GDs can spread through populations even if they reduce the ﬁtness of each carrier animal. This gives GDs more scope to escape the target population and unintentionally affect extraneous ecosystems. Even when new traits' direct impact on a target is understood, the drive may have side effects on the surroundings. A mutation could happen mid-drive, which has the potential to allow unwanted traits to "ride along". Cross-breeding or gene flow potentially allow a drive to move beyond its target population. With the progress in this space, the risks associated with current GD architecturesare likely to be reduced with the realizationof self-limiting GD strategies.
1. Noble et al. Current CRISPR gene drive systems are likely to be highly invasive in wild populations. e Life. 2018; 7:e33423.
2. Prowse et al. Dodging silver bullets: good CRISPR gene-drive design is critical for eradicating exotic vertebrates. Proc. R. Soc. B 284: 20170799.
3. McFarlane, D.J. et al. CRISPR-Based Gene Drives for Pest Control. Trends in Biotechnology, February 2018, Vol. 36, No. 2.
4. Champer et al. Novel CRISPR/Cas9 gene drive constructs reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations. PLo S Genet 13(7):e1006796.