Targeted Chromosomal Rearrangements via Combinatorial Use of CRISPR/Cas9 and Cre/LoxP Technologies in Caenorhabditis elegans.

Rearranged chromosomes have been applied to construct genetic balancers to manipulate essential genes in C. elegans. Although much effort has been put into constructing balancer chromosomes, approximately 6% (map units) of the C. elegans genome has not been covered, and this area lies mostly in pairing centers (PCs). Here, we developed a method for conditional chromosomal engineering through combinatorial use of the CRISPR/Cas9 and Cre/LoxP technologies. Functional DNA fragments containing LoxP sequences were inserted into designated genomic loci using a modified counterselection (cs)-CRISPR method. Then, heat-shock-induced Cre recombinase induced an inversion of the chromosomal region between the two LoxP sites. The chromosomal inversions were subsequently detected by the appearance of pharyngeal GFP. Through this method, we have successfully generated several chromosomal inversion lines, providing valuable resources for studying essential genes in pairing centers.

重排染色体已被用于构建遗传平衡子，以操纵秀丽隐杆线虫中的必需基因。尽管在构建平衡染色体方面已付出了很多努力，但秀丽隐杆线虫基因组中约6%（图距单位）尚未被覆盖，且该区域主要位于配对中心（PCs）。在此，我们开发了一种通过组合使用CRISPR/Cas9和Cre/LoxP技术进行条件性染色体工程的方法。使用改良的反向选择（cs）-CRISPR方法将含有LoxP序列的功能性DNA片段插入指定的基因组位点。然后，热激诱导的Cre重组酶诱导两个LoxP位点之间的染色体区域发生倒位。随后通过咽部绿色荧光蛋白（GFP）的出现来检测染色体倒位。通过这种方法，我们已成功产生了几条染色体倒位品系，为研究配对中心的必需基因提供了有价值的资源。