Emergent CRISPR–Cas-based technologies for engineering non-model bacteria

Volke, Daniel C.; Orsi, Enrico; Nikel, Pablo I.

doi:10.1016/j.mib.2023.102353

Cited by 25 publications

(17 citation statements)

References 86 publications

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“…CRISPR-mediated base-editing was introduced in bacteria only recently, yet these technologies have been rapidly adapted for diverse objectives, encompassing both fundamental and applied research (Cho et al, 2018;Volke et al, 2023). While the PAM requirements of CRISPR systems help mediating sequence-specific adaptive immunity in bacteria, the strict nature of PAM recognition constrains genome engineering applications (Adli, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…Precision genome editing continues to revolutionize life sciences, with a direct impact in multiple fields (Wang et al, 2022;Wang and Doudna, 2023)-including both fundamental studies and applied sciences, e.g. metabolic engineering (Cho et al, 2018;Zhao et al, 2021;Volke et al, 2023). Among these emerging approaches, base-editing of bacterial genomes assisted by clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas), allows for single-nucleotide-resolution modification of genomic sites in a homologous recombination-independent fashion (Anzalone et al, 2020;Nishida and Kondo, 2020;Sun et al, 2020;Walton et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

Kozaeva

Nielsen

Nikel

2023

Preprint

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Pseudomonasspecies are platforms for chemical production. To unleash their potential as cell factories, we designed synthetic biology tools based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) base-editors. A cytidine base-editor (CBE) and an adenine base-editor (ABE) were tailored for high-resolution genome engineering. CBE involves a cytidine deaminase fused to a nickase Cas9 fromStreptococcus pyogenes(SpnCas9) variant that converts cytidine to uracil, resulting in a C→T (or G→A) substitution. ABE relies on an adenine deaminase fused to a nickaseSpnCas9 variant to enable A→G (or T→C) editing. We established plasmid-borne, base-editing systems for efficient nucleotide substitution using a novel protospacer adjacent motif (PAM)-relaxedSpnCas9 (SpRY) variant. Base-editing was demonstrated by introducing prematureSTOPcodons in target genes (encoding both fluorescent proteins and metabolic functions), resulting in functional knockouts (using CBE). The wild-type genotype was recovered by eliminating the engineeredSTOPcodons (using ABE).P.putidacould be base-edited at ca. 75-95% efficiency for both types of editors. Furthermore, a set of induction-dependent vectors enabled efficient curing of the plasmid-based editing platform in a single, 8-h cultivation step. This strategy eases complex strain construction programs independently of homologous recombination and yields plasmid-free engineered cells.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

Kozaeva

Nielsen

Nikel

2023

Preprint

Self Cite

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“…[49] The domestication and engineering of non-model organism enabled the exploitation of emergent biotechnological processes contributing to bio-based economy. [50] The development of biomonitoring systems makes effective use of CRISPR/Cas technology in light of global climate change, increased eutrophication, the incidence of harmful algal blooms (HAB) and cyanobacterial harmful algal blooms (CHAB). [51] CRISPR/Cas gene editing technology have led to improve various morphological and nutritional traits of plants.…”

Section: Application Of Crispr/cas For Crop Improvementmentioning

confidence: 99%

CRISPR/Cas system: A revolutionary tool for crop improvement

Mishra,

Pandey

2024

Biotechnology Journal

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World's population is elevating at an alarming rate thus, the rising demands of producing crops with better adaptability to biotic and abiotic stresses, superior nutritional as well as morphological qualities, and generation of high‐yielding varieties have led to encourage the development of new plant breeding technologies. The availability and easy accessibility of genome sequences for a number of crop plants as well as the development of various genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs) has opened up possibilities to develop new varieties of crop plants with superior desirable traits. However, these approaches has limitation of being more expensive as well as having complex steps and time‐consuming. The CRISPR/Cas genome editing system has been intensively studied for allowing versatile target‐specific modifications of crop genome that fruitfully aid in the generation of novel varieties. It is an advanced and promising technology with the potential to meet hunger needs and contribute to food production for the ever‐growing human population. This review summarizes the usage of novel CRISPR/Cas genome editing tool for targeted crop improvement in stress resistance, yield, quality and nutritional traits in the desired crop plants.

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“…However, its protospacer adjacent motif (PAM) has the highly permissive NGG sequence, and off-targeting could occur . CRISPRi/spdCas9 circuits such as logic gates, waveform generators, and memories have been previously implemented, ,,− and applications in metabolic engineering, control of cellular phenotypes, biocontainment, and bioproduction have been demonstrated. − …”

Section: Introductionmentioning

confidence: 99%

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

De Marchi,

Shaposhnikov,

Gobaa

et al. 2024

ACS Synth. Biol.

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Synthetic circuit design is crucial for engineering microbes that process environmental cues and provide biologically relevant outputs. To reliably scale-up circuit complexity, the availability of parts toolkits is central. Streptococcus pyogenes (sp)-derived CRISPR interference/dead-Cas9 (CRISPRi/spdCas9) is widely adopted for implementing programmable regulations in synthetic circuits, and alternative CRISPRi systems will further expand our toolkits of orthogonal components. Here, we showcase the potential of CRISPRi using the engineered dCas9 from Staphylococcus aureus (sadCas9), not previously used in bacterial circuits, that is attractive for its low size and high specificity. We designed a collection of ∼20 increasingly complex circuits and variants in Escherichia coli, including circuits with static function like one-/two-input logic gates (NOT, NAND), circuits with dynamic behavior like incoherent feedforward loops (iFFLs), and applied sadCas9 to fix a T7 polymerase-based cascade. Data demonstrated specific and efficient target repression (100-fold) and qualitatively successful functioning for all circuits. Other advantageous features included low sadCas9-borne cell load and orthogonality with spdCas9. However, different circuit variants showed quantitatively unexpected and previously unreported steady-state responses: the dynamic range, switch point, and slope of NOT/NAND gates changed for different output promoters, and a multiphasic behavior was observed in iFFLs, differing from the expected bell-shaped or sigmoidal curves. Model analysis explained the observed curves by complex interplays among components, due to reporter geneborne cell load and regulator competition. Overall, CRISPRi/sadCas9 successfully expanded the available toolkit for bacterial engineering. Analysis of our circuit collection depicted the impact of generally neglected effects modulating the shape of component dose−response curves, to avoid drawing wrong conclusions on circuit functioning.

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Emergent CRISPR–Cas-based technologies for engineering non-model bacteria

Cited by 25 publications

References 86 publications

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

CRISPR/Cas system: A revolutionary tool for crop improvement

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

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