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 generations 1,2,3 . The question arises: if an active genetic terror event is released into a population, could we prevent the total spread of the undesired allele 4 ? Thus far, MCR protection methods require knowledge of the terror locus 5 . 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 locus 6 . 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 gRNA1 7 . We demonstrate the proof-of-concept and efficacy of our protection construct through a Graphical Markov model and computer simulation.