Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

Hao, Yile; Wang, Qinhua; Li, Jie; Yang, Shihui; Zheng, Yanli; Peng, Wenfang

doi:10.1098/rsob.210241

Cited by 12 publications

(7 citation statements)

References 55 publications

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“…Possibly, the Cas9 nuclease per se is toxic to the host cells. Similar observations have been reported for other bacteria (Zheng et al, 2020;Hao et al, 2022). Interesting, we found that the chromosome of P. alni Shahu encodes a native CRISPR-Cas system.…”

Section: Discussionsupporting

confidence: 91%

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu

Wang

Yang

et al. 2022

Front. Microbiol.

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Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.

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Section: Discussionsupporting

confidence: 91%

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu

Wang

Yang

et al. 2022

Front. Microbiol.

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“…In particular, the use of the Cas3 K39A variant in combination with a donor template enhanced editing efficiency. Recently, a Cas3 helicase variant of the Zymomonas mobilis type I-F system was applied endogenously to carry out genome editing with high efficiency (46). In that study, crRNA was used to target each strand of the target, resulting in "dual nicking" of the target gene.…”

Section: Discussionmentioning

confidence: 99%

Repurposing the atypical Type I-G CRISPR system for bacterial genome engineering

Shangguan

White

2023

Preprint

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The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilise a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.

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“…In Z. mobilis , gene deletion and insertion and single base substitution can be realized with 100% efficiency, but deletion of large genomic fragments such as a 10-kb fragment was only achieved with less than 50% efficiency [80] . To increase the deletion efficiency for large genomic fragments, a recent study created an nCas3 variant by replacing the catalytic residues with alanine and showed that the I-F Cascade-nCas3 enabled gene insertion, gene deletion, single base substitution and deletion of one or even two large genomic fragments at once with almost 100% efficiency [81] .…”

Section: Type I Crispr-cas-mediated Gene Editingmentioning

confidence: 99%

Type I CRISPR-Cas-mediated microbial gene editing and regulation

Xu,

Chen,

et al. 2023

AIMSMICRO

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<abstract> <p>There are six major types of CRISPR-Cas systems that provide adaptive immunity in bacteria and archaea against invasive genetic elements. The discovery of CRISPR-Cas systems has revolutionized the field of genetics in many organisms. In the past few years, exploitations of the most abundant class 1 type I CRISPR-Cas systems have revealed their great potential and distinct advantages to achieve gene editing and regulation in diverse microorganisms in spite of their complicated structures. The widespread and diversified type I CRISPR-Cas systems are becoming increasingly attractive for the development of new biotechnological tools, especially in genetically recalcitrant microbial strains. In this review article, we comprehensively summarize recent advancements in microbial gene editing and regulation by utilizing type I CRISPR-Cas systems. Importantly, to expand the microbial host range of type I CRISPR-Cas-based applications, these structurally complicated systems have been improved as transferable gene-editing tools with efficient delivery methods for stable expression of CRISPR-Cas elements, as well as convenient gene-regulation tools with the prevention of DNA cleavage by obviating deletion or mutation of the Cas3 nuclease. We envision that type I CRISPR-Cas systems will largely expand the biotechnological toolbox for microbes with medical, environmental and industrial importance.</p> </abstract>

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Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

Cited by 12 publications

References 55 publications

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu

Repurposing the atypical Type I-G CRISPR system for bacterial genome engineering

Type I CRISPR-Cas-mediated microbial gene editing and regulation

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