Application of different types of CRISPR/Cas-based systems in bacteria

Liu, Zhenquan; Dong, Huina; Cui, Yali; Liu, Cong; Zhang, Dawei

doi:10.1186/s12934-020-01431-z

Cited by 130 publications

(97 citation statements)

References 121 publications

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“…We successfully applied our framework to multiple base strains of S. cerevisiae (i.e., BY4741, CEN.PK, Sigma) for rapid biosynthesis of native and heterologous chemical products from 3 distinct branches of metabolism as a proof of concept. The broad applicability of CRISPR/dCas9 modulation across species 76 , 77 and the growing repertoire of protocols for cell extract preparation from diverse organisms 59 , 75 , 78 present an immense application space of rewired metabolism in cell-free contexts. This could include increasing the productivity of established E. coli CFME platforms for high-value molecules 30 , 79 , 80 and materials 38 , 81 with the potential for scaling up cell-free reactions 31 , 82 .…”

Section: Discussionmentioning

confidence: 99%

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Rasor

Brown

et al. 2021

Nat Commun

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Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.

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Section: Discussionmentioning

confidence: 99%

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Rasor

Brown

et al. 2021

Nat Commun

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“…Accounting for their nature of simplicity, the types involved under Class II Classification of CRISPR/Cas are reported to be the easiest in using for effective genome edited and manipulation of nucleic acids devoid of cells. Apart from being involved in gene editing the types of CRISPR/Cas systems derived from Class2 classification i. e., type II, V and VI are said to be instrumental in developing competent diagnostic platform of disease detection [36]. Brief representation of the fore said classification along with their application pertaining to class II CRISPR/Cas is as follows, Figure 2.…”

Section: Types Of Crispr/cas Systems and Their Applicationsmentioning

confidence: 99%

“…The dCas9 system is designed with modification in the basic activity of the conventional Cas9 protein in terms of deactivating the nucleic acid cleavage potential and only retaining the specific binding ability to target dsDNA. This was accomplished by inducing two point mutations H840A and D10A in the HNH and RuvC nuclease domain of the conventional Cas9 effector protein [36,53].…”

Section: Crispr In Diagnosismentioning

confidence: 99%

CRISPR Cas/Exosome Based Diagnostics: Future of Early Cancer Detection

Mubthasima¹,

Pande²,

Prakash³

et al. 2022

Rural Health

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Trending and Thriving, CRISPR/Cas has expanded its wings towards diagnostics in recent years. The potential of evading off targeting has not only made CRISPR/Cas an effective therapeutic aid but also an impressive diagnostic tool for various pathological conditions. Exosomes, 30 - 150nm sized extracellular vesicle present and secreted by almost all type of cells in body per se used as an effective diagnostic tool in early cancer detection. Cancer being the leading cause of global morbidity and mortality can be effectively targeted if detected in the early stage, but most of the currently used diagnostic tool fails to do so as they can only detect the cancer in the later stage. This can be overcome by the use of combo of the two fore mentioned diagnostic aids, CRISPR/Cas alongside exosomes, which can bridge the gap compensating the cons. This chapter focus on two plausible use of CRISPR/Cas, one being the combinatorial aid of CRISPR/Cas and Exosome, the two substantial diagnostic tools for successfully combating cancer and other, the use of CRISPR in detecting and targeting cancer exosomes, since they are released in a significant quantity in early stage by the cancer cells.

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“…Thus far, the Cas12a orthologs are shown to be able to mediate genome editing in human cells (Zetsche et al 2015 ; Tóth et al 2018 ). In recent years, Cas12a has also been employed for genome editing and gene regulation in bacteria, although the information is very limited compared to its application in eukaryotes (Yao et al 2018 ; Adiego-Pérez et al 2019 ; Liu et al 2020 ). Among the three variants, FnCas12a has been used the most to facilitate genetic engineering in bacteria (Table 2 ).…”

Section: Crispr-cas12a As An Attractive System For Genetic Engineeringmentioning

confidence: 99%

Recent advances of Cas12a applications in bacteria

Meliawati

Schilling

Schmid

2021

Appl Microbiol Biotechnol

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Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches

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Application of different types of CRISPR/Cas-based systems in bacteria

Cited by 130 publications

References 121 publications

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

CRISPR Cas/Exosome Based Diagnostics: Future of Early Cancer Detection

Recent advances of Cas12a applications in bacteria

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