Expanding targeting scope, editing window, and base transition capability of base editing in <i>Corynebacterium glutamicum</i>

Wang, Yu; Liu, Ye; Li, Junwei; Yang, Yi; Ni, Xiaomeng; Cheng, Haijiao; Huang, Teng; Guo, Yanmei; Ma, Hongwu; Zheng, Ping; Wang, Meng; Sun, Jibin; Ma, Yanhe

doi:10.1002/bit.27121

Cited by 45 publications

(47 citation statements)

References 41 publications

(107 reference statements)

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“…The low efficiency of triple-site editing could be possibly caused by the lower amount of the base editor at each locus than those targeting single loci. And a developed system with expanding targeting scope, editing window, and base transition capability was further constructed in C. glutamicum by the same group ( Wang et al, 2019 ).…”

Section: Regulation Of Gene Expression By Crispr/cas Toolboxmentioning

confidence: 99%

Development and Application of CRISPR/Cas in Microbial Biotechnology

Ding

Yang

Shi

2020

Front. Bioeng. Biotechnol.

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The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has been rapidly developed as versatile genomic engineering tools with high efficiency, accuracy and flexibility, and has revolutionized traditional methods for applications in microbial biotechnology. Here, key points of building reliable CRISPR/Cas system for genome engineering are discussed, including the Cas protein, the guide RNA and the donor DNA. Following an overview of various CRISPR/Cas tools for genome engineering, including gene activation, gene interference, orthogonal CRISPR systems and precise single base editing, we highlighted the application of CRISPR/Cas toolbox for multiplexed engineering and high throughput screening. We then summarize recent applications of CRISPR/Cas systems in metabolic engineering toward production of chemicals and natural compounds, and end with perspectives of future advancements.

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Section: Regulation Of Gene Expression By Crispr/cas Toolboxmentioning

confidence: 99%

Development and Application of CRISPR/Cas in Microbial Biotechnology

Ding

Yang

Shi

2020

Front. Bioeng. Biotechnol.

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“…After the base editing procedure, DNA sequencing showed that spacers HexR ( Supplementary Figure S5 ), PA2018 ( Figure 4A ), HexR-2 and HexR-3 ( Figure 5B ) exhibited significantly higher mutation efficiencies than the original spacers HexR-5, PA2018-2, and HexR-4. To minimize the motif preference of APOBEC1 or the disruption of off-target effects, the design of the 20-bp spacer with a NGG or NG PAM for gene inactivation should be assessed by base editing designing tools ( Xiao et al, 2014 ; Hwang et al, 2018 ; Wang et al, 2019 ) in an effort to overcome this potential limitation ( Wang et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

“…The element UGI was fused to the C-terminus of APOBEC1-XTEN- eSpCas9pp D10A in pSEVA-Module 4, giving rise to pSEVA-Module 6. pSEVA-Module 6 was named pSEVA6BE, which was used as the template for the construction of following base editing plasmids ( Supplementary Table S2 ). All of the base editing spacers ( Supplementary Table S4 ) used in this study were designed and analyzed by CasOT ( Xiao et al, 2014 ), gBIG ( Wang et al, 2019 ), or BE-Designer ( Hwang et al, 2018 ).…”

Section: Methodsmentioning

confidence: 99%

“…To circumvent reversal of this base conversion by UNG, a bacteriophage derived uracil DNA glycosylase inhibitor (UGI) is usually fused to the C-terminus of the APOBEC1-nCas9 D10A complex to inhibit UNG and improve base editing efficiencies. Currently, cytidine deaminase-based base editing system have been extended to Escherichia coli (Nishida et al, 2016), Clostridium beijerinckii (Li et al, 2019), Klebsiella pneumonia (Yu et al, 2018), Corynebacterium glutamicum (Wang et al, 2019), and some Pseudomonas spp. (Chen et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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CRISPR-Assisted Multiplex Base Editing System in Pseudomonas putida KT2440

Sun

Liang

et al. 2020

Front. Bioeng. Biotechnol.

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Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9pp D10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25-100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9pp D10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90-100% and 25-35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.

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“…Therefore, the cas9 copy number has been reduced through genomic integration, and weak promoters including P tuf have been used to develop the genome editing tool of C. glutamicum [12]. A modi ed Cas9 recognizing the 5′-NG sequence as the PAM sequence has been recently reported to recognize more target DNA sequences during genome engineering in C. glutamicum [23].…”

Section: Introductionmentioning

confidence: 99%

Efficient and Accurate Single-Base Genome Editing in Corynebacterium Glutamicum by Target-Mismatched CRISPR/Cpf1

Kim¹,

Oh²,

Lee³

2020

Preprint

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Background CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in C. glutamicum.Results This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in Corynebacterium glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5–95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were accurately edited.Conclusions The present results indicate that the target-mismatched Cpf1 negative selection method is not only efficient in C. glutamicum, but also an accurate single-base genome editing method.

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Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

Cited by 45 publications

References 41 publications

Development and Application of CRISPR/Cas in Microbial Biotechnology

Development and Application of CRISPR/Cas in Microbial Biotechnology

CRISPR-Assisted Multiplex Base Editing System in Pseudomonas putida KT2440

Efficient and Accurate Single-Base Genome Editing in Corynebacterium Glutamicum by Target-Mismatched CRISPR/Cpf1

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