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 (Esvelt et al., 2014, Gantz and Bier, 2015, Gantz and Bier, 2016). The question arises: if an active genetic terror event is released into a population, could we prevent the total spread of the undesired allele (Gantz, et al., 2015, Webber et al., 2015)? Thus far, effective protection methods require knowledge of the terror locus (Grunwald et al., 2019). 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 (Hammond et al., 2017). 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 (Khan, 2016, Zetsche et al., 2015). We demonstrate the proof-of-concept and efficacy of our protection construct through a Graphical Markov model and computer simulation.

诱变链反应 (MCR) 是一种使用 CRISPR-Cas 构建体引入归巢核酸内切酶的遗传工具，允许基因驱动以最少的世代影响整个种群（Esvelt 等人，2014 年；Gantz 和 Bier，2015 年） ，甘茨和比尔，2016 年）。问题出现了：如果一个活跃的遗传恐怖事件被释放到一个群体中，我们能否阻止不需要的等位基因的全面传播（Gantz 等，2015；Webber 等，2015）？迄今为止，有效的保护方法需要了解恐怖分子的位置（Grunwald 等，2019）。在这里，我们介绍了一种新方法，未知位点的自催化保护(a-PUL)，其目的是在没有任何恐怖修饰轨迹的先验知识的情况下，在人群中传播并阻止和减少活跃的恐怖事件的传播，从而允许以后的自然选择和 ERACR 驱动恢复正常轨迹（Hammond 等等，2017）。a-PUL，使用诱变链反应，包括 (i) 编码非 Cas9 核酸内切酶的片段，能够进行同源定向修复，建议为 II 型核酸内切酶 Cpf1 (Cas12a)，(ii) 普遍表达的编码 gRNA 的基因（ gRNA1) 具有对 Cpf1 特异的 U4AU4 3'-突出端和对某些所需的非编码 DNA 基因组序列特异的 crRNA，(iii) 一个普遍表达的基因，编码两种 gRNA (gRNA2/gRNA3)，两者都具有特异于 Cas9 的 tracrRNA 和对 Cas9 基因座的两个不同位点特异的 crRNA，(iv) 位于 Cpf1/gRNA1/gRNA2/gRNA3 盒两侧的同源臂，与由 gRNA1 引导的目标切割周围区域相同（Khan，2016，Zetsche 等，2015）。我们通过图形马尔可夫模型和计算机模拟证明了我们保护构造的概念验证和有效性。