Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus
Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus
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DOI：
10.1093/femsle/fnz105
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发表时间：
2019-05-01
影响因子：
2.1
通讯作者：
Barrangou, Rodolphe
中科院分区：
文献类型：
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作者：
Canez, Cassandra;Selle, Kurt;Barrangou, Rodolphe
CRISPR-Cas系统通过DNA编码、RNA介导、核酸酶依赖性靶向和切割提供针对原核生物中噬菌体的适应性免疫。由于细菌中 DNA 修复途径效率低下且相对有限，CRISPR-Cas 系统可以重新用于选择序列变异的致命 DNA 靶向。在本研究中，评估了模型生物嗜热链球菌 DGCC7710 中内源 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 型系统的染色体靶向的遗传和表型结果。当递送 0.4-400 ng 质粒 DNA 时，使用这两个系统均观察到以剂量依赖性方式有效杀伤。使用靶向 PCR 筛选和基因组测序来确定能够生存的遗传基础，表明 I-E 型自靶向的逃避主要是切除靶向间隔区的低频缺陷质粒的结果。从基因组位点 lacZ 的 II-A 型靶向中恢复的最显着的基因型是源自两个独立 galE 编码区中相同保守序列之间同源重组 (HR) 的 34 kb 缺失，导致基因组损失 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，从而导致 CRISPR-Cas 系统逃避基因组靶向。
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.