Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays

@article{Reis2019SimultaneousRO,
  title={Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays},
  author={Alexander C. Reis and Sean M. Halper and Grace E. Vezeau and Daniel P. Cetnar and Ayaan Hossain and Phillip R. Clauer and H. Salis},
  journal={Nature Biotechnology},
  year={2019},
  volume={37},
  pages={1294-1301}
}
Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop… Expand
dCas9 regulator to neutralize competition in CRISPRi circuits
TLDR
The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes, and allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits. Expand
Automated design of thousands of nonrepetitive parts for engineering stable genetic systems
TLDR
The Nonrepetitive Parts Calculator is developed to rapidly generate thousands of highly nonrepetive genetic parts from specified design constraints, including promoters, ribosome-binding sites and terminators, and it is shown that using nonrePETitive genetic parts substantially reduces homologous recombination, resulting in greater genetic stability. Expand
A multiplex CRISPR interference tool for virulence gene interrogation in an intracellular pathogen
TLDR
Using crRNAs directed against virulence protein-encoding genes, it is demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISpri fully recapitulated the growth defect of deletion strains. Expand
Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.
TLDR
A modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISpri activity is developed and its broad applicability is demonstrated. Expand
An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida
TLDR
A single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system is presented, which enables tunable, tightly controlled gene repression of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. Expand
Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition.
TLDR
A simple and robust method to knock-out multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs is designed, exemplifying the potency of this approach by targeting the well-characterised acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family. Expand
A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila
TLDR
A multiplex CRISPR interference tool for Legionella pneumophila, the intracellular pathogen responsible for Legionnaires’ disease, that enables precise interrogation of multiple virulence genes simultaneously during host infection and holds great promise for probing large sets of genes in bulk. Expand
Efficient Multiplex Gene Repression by CRISPR-dCpf1 in Corynebacterium glutamicum
TLDR
The CRISPR-dCpf1-based multiplex gene repression system was developed and holds promise for metabolic engineering of C. glutamicum and was applied to repress four genes involved in lysine biosynthesis with a single array. Expand
One-shot generation of duodecuple (12x) mutant Arabidopsis: Highly efficient routine editing in model species
TLDR
A toolkit based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants is presented. Expand
Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi
TLDR
Detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest are provided. Expand
...
1
2
3
4
5
...

References

SHOWING 1-10 OF 49 REFERENCES
CRISPR interference-guided multiplex repression of endogenous competing pathway genes for redirecting metabolic flux in Escherichia coli
TLDR
The tunable CRISPRi system is demonstrated to be a robust platform for multiplex modulation of endogenous gene expression that can be used to enhance biosynthetic pathway productivity, with n-butanol as the test case. Expand
Orthologous CRISPR-Cas9 enzymes for Combinatorial Genetic Screens
TLDR
The “Big Papi” approach described here will be widely applicable for the study of combinatorial phenotypes and generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types. Expand
CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces.
TLDR
A novel CRISPRi system for multiplex gene repression in the model strain Streptomyces coelicolor is developed and successfully employed for functional gene screening, and an orphan response regulator (RR) containing an RNA-binding ANTAR domain is identified being involved in bacterial growth. Expand
Enabling Genetic Analysis of Diverse Bacteria with Mobile-CRISPRi
TLDR
‘Mobile-CRISpri’ is established, a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation that enables genetic investigations in non-model bacteria. Expand
A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation
TLDR
This work develops and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9- based binding and Cas 9-based cleavage at all DNA sites. Expand
Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks
TLDR
This work constructed a set of NOT gates by designing five synthetic Escherichia coli σ70 promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. Expand
A Model for Resource Competition in CRISPR-Mediated Gene Repression
TLDR
This work develops a general model that takes into account the sharing of a limited amount of dCas9 protein for arbitrary CRISPR-mediated gene repression networks, and demonstrates that perfect adaptation to resource fluctuations can be achieved in cascades with an even number of repressors. Expand
tCRISPRi: tunable and reversible, one-step control of gene expression
TLDR
The results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries. Expand
Antisense transcription as a tool to tune gene expression
TLDR
This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering. Expand
Programming cells by multiplex genome engineering and accelerated evolution
TLDR
The multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties by facilitating rapid and continuous generation of a diverse set of genetic changes. Expand
...
1
2
3
4
5
...