Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.
Functional Genomics Graduate Program, North Carolina State University, Raleigh, NC 27695, USA.
FEMS Microbiol Lett. 2019 May 1;366(9). doi: 10.1093/femsle/fnz105.
CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems.
CRISPR-Cas 系统通过 DNA 编码、RNA 介导、核酸酶依赖的靶向和切割，为原核生物提供针对噬菌体的适应性免疫。由于细菌中低效和相对有限的 DNA 修复途径，CRISPR-Cas 系统可以被重新用于致命的 DNA 靶向，从而选择序列变体。在这项研究中，评估了模型生物嗜热链球菌 DGCC7710 中内源性 I 型和 II 型 CRISPR-Cas 系统的相对杀伤效率。此外，还分析了质粒编程的 I-E 型或 II-A 型系统对染色体靶向的遗传和表型结果。当递送 0.4-400ng 质粒 DNA 时，两种系统都以剂量依赖性方式观察到有效的杀伤。靶向 PCR 筛选和基因组测序用于确定使生存成为可能的遗传基础，表明 I-E 型自我靶向的逃避主要是由于低频缺陷质粒切除靶向间隔子所致。从 lacZ 基因组定位的 II-A 靶向中恢复的最显著基因型是源自两个单独 galE 编码区中相同保守序列之间同源重组 (HR) 的 34kb 缺失，导致基因组丢失 2%。总的来说，这些结果表明 HR 有助于细菌基因组的可塑性和重塑，从而逃避 CRISPR-Cas 系统对基因组的靶向。
