Howard Hughes Medical Institute and School of Biological Sciences, University of Utah, Salt Lake City, Utah, United States of America.
PLoS Genet. 2021 Nov 8;17(11):e1009755. doi: 10.1371/journal.pgen.1009755. eCollection 2021 Nov.
Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50-100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.
使用基于质粒的 CRISPR 试剂在秀丽隐杆线虫中进行基因编辑需要对许多动物进行微注射才能产生单个编辑。质粒携带的 Cas9 的生殖系沉默是编辑效率低下的主要原因。在这里，我们提供了一组秀丽隐杆线虫品系，它们从整合的转基因中在生殖系中组成性表达 Cas9。这些品系显着提高了基于质粒的 CRISPR 编辑的成功率。对于简单的、短同源臂 GFP 插入，根据靶基因座，50-100%的注射动物通常会产生编辑后的后代。从染色体外阵列进行模板引导的编辑可以维持多代。我们已经在多个染色体上构建了带有 Cas9 转基因的菌株。此外，每个 Cas9 基因座还包含一个热休克驱动的 Cre 重组酶，用于选择性标记物的去除和明亮的荧光标记物，便于杂交。这些整合的 Cas9 菌株大大减少了产生单个基因组编辑的工作量。
