Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.
Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.
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DOI：
10.1038/s42003-023-04994-w
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发表时间：
2023-06-09
影响因子：
5.9
通讯作者：
Hooven, Thomas A.
中科院分区：
文献类型：
--
作者：
Gopalakrishna, Kathyayini P.;Hillebrand, Gideon H.;Bhavana, Venkata H.;Elder, Jordan L.;D'Mello, Adonis;Tettelin, Herve;Hooven, Thomas A.
B族链球菌（GBS; S.无乳症）引起绒毛膜炎、新生儿败血症，并且还可在健康或免疫受损的成人中引起疾病。GBS具有II-A型CRISPR-Cas9系统，可以防御细菌细胞内的外源DNA。最近的几篇出版物表明，GBS Cas9通过与其作为特异性RNA可编程核酸内切酶的功能解偶联的机制影响全基因组转录。我们通过产生具有特定功能缺陷的几种同基因变体来检查GBS Cas9对全基因组转录的影响。我们比较了来自Δcas9 GBS的全基因组RNA-seq与全长Cas9基因缺失; dcas 9在切割DNA的能力上有缺陷，但仍然能够结合频繁出现的原型间隔区相邻基序;以及保留其催化结构域但无法结合原型间隔区相邻基序的scas 9。将scas 9 GBS与其他变体进行比较，我们鉴定了非特异性前间区序列邻近基序结合作为GBS中全基因组Cas9转录效应的驱动因素。我们还表明，来自非特异性扫描的Cas9转录效应倾向于影响参与细菌防御和核苷酸或碳水化合物运输和代谢的基因。虽然通过下一代测序分析可以检测到全基因组转录效应，但它们不会导致脓毒症小鼠模型中的毒力变化。我们还证明，从GBS染色体表达的无催化活性的dCas 9可以与直接的、基于质粒的、单向导RNA表达系统一起使用，以抑制特定GBS基因的转录，而不会潜在地混淆脱靶效应。我们预计，这一系统将是有用的非必需和必需的基因在GBS的生理和发病机制的研究。Cas9在条件致病菌无乳链球菌中诱导各种转录作用，特别是与细菌防御、核苷酸或碳水化合物代谢相关。
Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δcas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis. Cas9 induces various transcriptional effects in the opportunistic pathogen, Streptococcus agalactiae, especially related to bacterial defense, nucleotide or carbohydrate metabolism.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
13.2
作者：
Dong Y;Ma K;Cao Q;Huang H;Nie M;Liu G;Jiang M;Lu C;Liu Y
通讯作者：
Liu Y
影响因子：
3.2
作者：
Abranches, Jacqueline;Nascimento, Marcelle M.;Burne, Robert A.
通讯作者：
Burne, Robert A.