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

Kozaeva, Ekaterina; Nielsen, Zacharias S.; Nikel, Pablo I.

doi:10.1101/2023.04.16.537106

Cited by 3 publications

(2 citation statements)

References 83 publications

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“…Pablo Casasso has set a new benchmark in CRISPR-Cas technology by developing a toolkit that enables accurate and reversible DNA modifications in bacteria, thus method goes beyond the limitations of traditional CRISPR technology and significantly enhances industry and research capabilities to create bacterial cell factories. pAblopCasso facilitates the development of bacteria for various bio production applications, including pharmaceuticals and biofuels, aligned with sustainable production objectives, by enabling quick and accurate editing Ekaterina [38].…”

Section: Visionary Concepts Of Crispr/cas9 Technologymentioning

confidence: 99%

Evaluating CRISPR/Cas9's Progressive Role in Crop Enhancement and Sustainable Agriculture

Sathiyabalan,

Amir,

Sandhu

et al. 2024

J. Adv. Biol. Biotechnol.

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This review paper provides a comprehensive exploration of the progressive effect of CRISPR/Cas9 genome altering innovation on functional genomics and crop enhancement. It highlights the evolution of CRISPR technology, emphasizing its versatile applications in plant breeding. The paper discusses the transition from gene knockouts to precise modifications and multiplex genome engineering, with a focus on enhancing plant nutrition, disease resistance, and drought tolerance. Traditional Cas9-guide RNA delivery methods are compared with ascending CRISPR ribonucleoproteins (RNPs) as solutions to overcome limitations associated with plasmid-based systems. The booming worldwide claim for food crops underscores the possibility of CRISPR/Cas9-based genome editing for improving cereal features. Earlier innovations like zinc finger nucleases (ZFNs) and TALENs, CRISPR/Cas9 offers a more cost-effective and time-efficient approach. It enables the blueprint and cloning of numerous gRNAs for accurate genome targeting, with enhanced specificity and efficiency achieved through modified Cas9 cassettes and enzymes from various bacterial species. The review also emphasizes CRISPR/Cas9's potential to develop non-genetically modified (Non-GMO) crops with certain needed traits, thereby boosting yield potential under various stress conditions. Furthermore, the article discusses problems and solutions analogous with CRISPR/Cas9-based genome editing, providing valuable insights for researchers in crop advancement.

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Section: Visionary Concepts Of Crispr/cas9 Technologymentioning

confidence: 99%

Evaluating CRISPR/Cas9's Progressive Role in Crop Enhancement and Sustainable Agriculture

Sathiyabalan,

Amir,

Sandhu

et al. 2024

J. Adv. Biol. Biotechnol.

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“…It appears that there is potential for further optimization. Recently, cytidine deaminase‐based toolsets that enable efficient multiplex editing in P. putida have been developed (Kozaeva et al., 2024; Volke et al., 2022; Yue et al., 2022). However, this approach necessitates several prerequisites and can potentially introduce unknown mutations beyond the spacer region (Volke et al., 2022).…”

Section: Introductionmentioning

confidence: 99%

Establishing a straightforward I‐SceI‐mediated recombination one‐plasmid system for efficient genome editing in P. putidaKT2440

Meng,

Köbbing,

Blank

2024

Microbial Biotechnology

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Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever‐improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one‐plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I‐SceI expressing plasmid pSW‐2 as a counterselection marker to accelerated plasmid curing; (2) double‐strand break introducing gene I‐sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG‐RIS. The single plasmid of pEMG‐RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I‐SceI encoding gene. Here, the usability of the pEMG‐RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.

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One-for-all gene inactivation via PAM-independent base editing in bacteria

Li,

Wei,

Wang

et al. 2024

Preprint

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Base editing is preferable for bacterial gene inactivation without generating double strand breaks, requiring homology recombination or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif (PAM). Herein, we report an unconstrained base editing system to enable the inactivation of any genes of interests (GOIs) in bacteria. We first employed a dCas9 derivative, dSpRY, as the effector to build a base editor with activation-induced cytidine deaminase, releasing the dependence on PAM. Then, we programmed the base editor to exclude the START codon of a GOI instead of introducing STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms inEscherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. The effectiveness of XSTART was also demonstrated in probioticE. coliNissle 1917 and photoautotrophic cyanobacteriumSynechococcus elongatus, illustrating its potential in reprogramming clinically and industrially relevant chassis. To be noticed, we observed a relatively high frequency of off-target events as a trade-off for the efficacy and universality.

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

Cited by 3 publications

References 83 publications

Evaluating CRISPR/Cas9's Progressive Role in Crop Enhancement and Sustainable Agriculture

Evaluating CRISPR/Cas9's Progressive Role in Crop Enhancement and Sustainable Agriculture

Establishing a straightforward I‐SceI‐mediated recombination one‐plasmid system for efficient genome editing in P. putidaKT2440

One-for-all gene inactivation via PAM-independent base editing in bacteria

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