Autocatalytic–Protection for an Unknown Locus CRISPR–Cas Countermeasure for Undesired Mutagenic Chain Reactions

The mutagenic chain reaction (MCR) is a genetic tool to use a CRISPR–Cas construct to introduce a homing endonuclease, allowing gene drive to influence whole populations in a minimal number of generations1,2,3. The question arises: if an active genetic terror event is released into a population, could we prevent the total spread of the undesired allele4? Thus far, MCR protection methods require knowledge of the terror locus5. Here we introduce a novel approach, an autocatalytic-Protection for an Unknown Locus (a-PUL), whose aim is to spread through a population and arrest and decrease an active terror event’s spread without any prior knowledge of the terror-modified locus, thus allowing later natural selection and ERACR drives to restore the normal locus6. a-PUL, using a mutagenic chain reaction, includes (i) a segment encoding a non-Cas9 endonuclease capable of homology-directed repair suggested as Type II endonuclease Cpf1 (Cas12a), (ii) a ubiquitously-expressed gene encoding a gRNA (gRNA1) with a U4AU4 3′-overhang specific to Cpf1 and with crRNA specific to some desired genomic sequence of non-coding DNA, (iii) a ubiquitously-expressed gene encoding two gRNAs (gRNA2/gRNA3) both with tracrRNA specific to Cas9 and crRNA specific to two distinct sites of the Cas9 locus, and (iv) homology arms flanking the Cpf1/gRNA1/gRNA2/gRNA3 cassette that are identical to the region surrounding the target cut directed by gRNA17. We demonstrate the proof-of-concept and efficacy of our protection construct through a Graphical Markov model and computer simulation.

Advances in Genetics Research

Genes are understandably crucial to physiology, morphology and biochemistry, but the idea of genes contributing to individual differences in behavior once seemed outrageous. Nevertheless, some scientists have aspired to understand the relationship between genes and behavior, and their research has become increasingly informative and productive over the past several decades. At the forefront of behavioral genetics research is the fruitfly Drosophila melanogaster, which has provided us with important insights into the molecular, cellular and evolutionary bases of behavior. By employing this development in their experiments with laboratory fruit flies, Gantz and Bier demonstrated that by arranging the standard components of this anti-viral defense system in a novel configuration, a mutation generated on one copy of a chromosome in fruit flies spreads automatically to the other chromosome. The end result, Bier says, is that both copies of a gene could be inactivated “in a single shot.” The two biologists call their new genetic method the “mutagenic chain reaction,” or MCR. “MCR is remarkably active in all cells of the body with one result being that such mutations are transmitted to offspring via the germline with 95 percent efficiency. Thus, nearly all gametes of an MCR individual carry the mutation in contrast to a typical mutant carrier in which only half of the reproductive cells are mutant.” Bier says “there are several profound consequences of MCR. First, the ability to mutate both copies of a gene in a single generation should greatly accelerate genetic research in diverse species. For example, to generate mutations in two genes at once in an organism is typically time consuming, because it requires two generations, and involved, because it requires genetic testing to identify rare progeny carrying both mutations. Now, one should simply be able to cross individuals harboring two different MCR mutants to each other and all their direct progeny should be mutant for both genes.”

Theoretical consequences of the Mutagenic Chain Reaction for manipulating natural populations

The use of recombinant genetic technologies for population manipulation has mostly remained an abstract idea due to the lack of a suitable means to drive novel gene constructs to high frequency in populations. Recently Gantz and Bier showed that the use of CRISPR/Cas9 technology could provide an artificial drive mechanism, the so-called Mutagenic Chain Reaction (MCR), which could lead to rapid fixation of even a deleterious introduced allele. We establish the equivalence of this system to models of meiotic drive and review the results of simple models showing that, when there is a fitness cost to the MCR allele, an internal equilibrium exists that is usually unstable. Introductions must be at a frequency above this critical point for the successful invasion of the MCR allele. These modeling results have important implications for application of MCR in natural populations.