Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

被引：101

作者：

Fontana, Jason ^{[1
]}

Dong, Chen ^{[2
]}

Kiattisewee, Cholpisit ^{[1
]}

Chavali, Venkata P. ^{[3
]}

Tickman, Benjamin, I ^{[1
]}

Carothers, James M. ^{[1
,3
,4
]}

Zalatan, Jesse G. ^{[2
,3
,4
]}

机构：

[1] Univ Washington, Mol Engn & Sci Inst, Seattle, WA 98195 USA

[2] Univ Washington, Dept Chem, Seattle, WA 98195 USA

[3] Univ Washington, Dept Chem Engn, Seattle, WA 98195 USA

[4] Univ Washington, Ctr Synthet Biol, Seattle, WA 98195 USA

来源：

NATURE COMMUNICATIONS | 2020年 / 11卷 / 01期

基金：

美国国家科学基金会;

关键词：

ESCHERICHIA-COLI; TRANSCRIPTIONAL ACTIVATION; SPACING REQUIREMENTS; RNA-POLYMERASE; LAC PROMOTER; DNA-BINDING; REPRESSION; PHASE; SOXS; ORIENTATION;

D O I：

10.1038/s41467-020-15454-y

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2-4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning similar to 40 bases and centered similar to 80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of sigma(70)-family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.

引用

页数：11

共 44 条

[1] Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system [J].