Engineering a <i>Streptococcus</i> Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Teng, Yuxi; Wang, Jian; Jiang, Tian; Zou, Yusong; Yan, Yajun

doi:10.1021/acssynbio.3c00366

Cited by 3 publications

(2 citation statements)

References 36 publications

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“…Green fluorescence (eGFP) was detected with an excitation wavelength of 485 nm and an emission wavelength of 528 nm, while red fluorescence with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The eGFP/OD600 or RFP/OD600 ratio was calculated as (fluorescence – background)/[(OD600 – background) × 1.76]. ,, All of the tests were performed using three biological repeats.…”

Section: Methodsmentioning

confidence: 99%

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Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor

Teng,

Gong,

Zhang

et al. 2024

ACS Synth. Biol.

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Transcription factor (TF)-based biosensors have arisen as powerful tools in the advancement of metabolic engineering. However, with the emergence of numerous bioproduction targets, the variety of applicable TF-based biosensors remains severely limited. In this study, we investigated and engineered an 1,2-propanediol (1,2-PD)-responsive transcription activator, PocR, from Salmonella typhimurium to enrich the current biosensor repertoire. Heterologous characterization of PocR in E. coli revealed a significantly limited operational range and dynamic range, primarily attributed to the leaky binding between PocR and its corresponding promoters in the absence of the 1,2-PD inducer. Promiscuity characterization uncovered the minor responsiveness of PocR toward glycerol and 1,2-butanediol (1,2-BD). Using AlphaFold-predicted structure and protein mutagenesis, we preliminarily explored the underlying mechanism of PocR. Based on the investigated mechanism, we engineered a PcoR-F46R/ G105D variant with an altered inducer specificity to glycerol, as well as a PocR-ARE (Q107A/S192R/A203E) variant with nearly a 4-fold higher dynamic range (6.7-fold activation) and a 20-fold wider operational range (0−20 mM 1,2-PD). Finally, we successfully converted PocR to a repressor through promoter engineering. Integrating the activation and repression functions established a versatile 1,2-PD-induced bifunctional regulation system based on PocR-ARE. Our work showcases the exploration and exploitation of an underexplored type of transcriptional activator capable of recruiting RNA polymerase. It also expands the biosensor toolbox by providing a 1,2-PD-responsive bifunctional regulator and glycerol-responsive activator.

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Section: Methodsmentioning

confidence: 99%

“…The eGFP/OD600 or RFP/OD600 ratio was calculated as (fluorescence – background)/[(OD600 – background) × 1.76]. 41 , 43 , 44 All of the tests were performed using three biological repeats.…”

Section: Methodsmentioning

confidence: 99%

Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor

Teng,

Gong,

Zhang

et al. 2024

ACS Synth. Biol.

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CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications

Burbano,

Kiattisewee,

Karanjia

et al. 2024

Annual Review of Chemical and Biomolecular Engineering

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In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

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Current Updates of CRISPR/Cas System and Anti-CRISPR Proteins: Innovative Applications to Improve the Genome Editing Strategies

Allemailem,

Almatroudi,

Alrumaihi

et al. 2024

IJN

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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated sequence (CRISPR/Cas) system is a cutting-edge genome-editing tool employed to explore the functions of normal and disease-related genes. The CRISPR/Cas system has a remarkable diversity in the composition and architecture of genomic loci and Cas protein sequences. Owing to its excellent efficiency and specificity, this system adds an outstanding dimension to biomedical research on genetic manipulation of eukaryotic cells. However, safe, efficient, and specific delivery of this system to target cells and tissues and their off-target effects are considered critical bottlenecks for the therapeutic applications. Recently discovered anti-CRISPR proteins (Acr) play a significant role in limiting the effects of this system. Acrs are relatively small proteins that are highly specific to CRISPR variants and exhibit remarkable structural diversity. The in silico approaches, crystallography, and cryo-electron microscopy play significant roles in elucidating the mechanisms of action of Acrs. Acrs block the CRISPR/Cas system mainly by employing four mechanisms: CRISPR/Cas complex assembly interruption, target-binding interference, target cleavage prevention, and degradation of cyclic oligonucleotide signaling molecules. Engineered CRISPR/Cas systems are frequently used in gene therapy, diagnostics, and functional genomics. Understanding the molecular mechanisms underlying Acr action may help in the safe and effective use of CRISPR/Cas tools for genetic modification, particularly in the context of medicine. Thus, attempts to regulate prokaryotic CRISPR/Cas surveillance complexes will advance the development of antimicrobial drugs and treatment of human diseases. In this review, recent updates on CRISPR/Cas systems, especially CRISPR/Cas9 and Acrs, and their novel mechanistic insights are elaborated. In addition, the role of Acrs in the novel applications of CRISPP/Cas biotechnology for precise genome editing and other applications is discussed.

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Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Cited by 3 publications

References 36 publications

Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor

Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor

CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications

Current Updates of CRISPR/Cas System and Anti-CRISPR Proteins: Innovative Applications to Improve the Genome Editing Strategies

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