gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing

Easmin, Farhana; Hassan, Naim; Sasano, Yu; Ekino, Keisuke; Taguchi, Hisataka; Harashima, Satoshi

doi:10.1016/j.jbiosc.2019.02.009

Cited by 22 publications

(19 citation statements)

References 13 publications

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“…Besides, large chromosomal fragment deletion methods were developed based on CRISPR/Cas9 system. Easmin developed a guide RNA-transient expression system (gRNA-TES), where two sgRNA expression fragments (locating to each end of target region on genome) and DNA donor containing CgLEU2 were co-transformed into host for a replacement of up to 500-kb regions with efficiencies of 67-100% ( Easmin et al, 2019 ).…”

Section: Application Of Crispr/cas System In Microbial Biotechnologymentioning

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: Application Of Crispr/cas System In Microbial Biotechnologymentioning

confidence: 99%

Development and Application of CRISPR/Cas in Microbial Biotechnology

Ding

Yang

Shi

2020

Front. Bioeng. Biotechnol.

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“…Some of the transformants constructed in previous study ) and transformants constructed in present study were used for spot test. We used the loxP site-deleted plasmid pSJ69 (Easmin et al 2019a) and pSJ70 (Easmin et al 2019b) derived from p3008 and p3009, respectively (Sugiyama et al 2005) as templates in which loxP-flanked DNA sequences were deleted to avoid undesired site-specific recombination. The plasmid pSJ69 harboring selective marker Candida glabrata LEU2 (CgLEU2) was used as a template to synthesize a DNA module for replacement of a particular chromosomal region.…”

Section: Strains Plasmids and Mediamentioning

confidence: 99%

“…Colony PCR and subsequent agarose gel electrophoresis were performed to check whether the expected replacement, splitting or duplication of the target chromosomal region had occurred in the transformants. Colony PCR was conducted according to Easmin et al (2019a). Primers for colony PCR used to check replacement, splitting and duplication are listed in Additional file 1: Table S2.…”

Section: Yeast Transformation Colony Pcrmentioning

confidence: 99%

Systematic approach for assessing whether undeletable chromosomal regions in Saccharomyces cerevisiae are required for cell viability

et al. 2020

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Previously, we identified 49 undeletable chromosomal regions harboring only non-essential genes in the genome of Saccharomyces cerevisiae. We proposed that there might be unknown synthetic lethal combinations of genes present in such undeletable regions of the genome. In this study, we chose four of the smallest undeletable chromosomal regions among the 49 and performed extensive further analyses to narrow down the gene-pairs responsible for lethality by replacing sub-regions in various combinations with a DNA module comprising the CgLEU2 marker. Although the methodology was different from previous study, interestingly the results revealed that not only the subregions but also the entire region was replaceable. To solve the apparent discrepancy between previous and present results, we further conducted additional analysis including investigation of suppressor mutation and mini-chromosome loss assay through the construction of mini-chromosome harboring two particular chromosomal regions with marked with URA3 marker by employing 5-FOA system. Based upon careful observation on the phenotype of colony formation on 5-FOA medium by spot test, we came to an important conclusion that particular chromosomal regions harboring only non-essential genes can be categorized into three classes, i.e., essential, non-essential and intrinsically essential. Intrinsically essential region is defined as appearance of papillae after mini-chromosome loss which implicates that the region is essential but compensatable against cell lethality. Our present study indicates that prudent and multiple approaches as performed in this study are needed to judge whether a particular chromosomal region of the S. cerevisiae genome is essential, non-essential or intrinsically essential but compensatable.

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“…This new technology is named CRISPR-PCDup SC medium lacking the appropriate amino acids was used for selection of transformants having the marker gene from the duplicating module. E. coli was transformed according to the method described by Easmin et al (2019a).…”

Section: Yeast and E Coli Transformationmentioning

confidence: 99%

“…Colony PCR was performed according to the method described by Easmin et al (2019a). Pulse field gel electrophoresis and Southern hybridization were performed according to Sasano et al (2016).…”

Section: Colony Pcr Pulse Field Gel Electrophoresis (Pfge) and Southmentioning

confidence: 99%

CRISPR-PCDup: a novel approach for simultaneous segmental chromosomal duplication in Saccharomyces cerevisiae

et al. 2020

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In our previous study, a novel genome engineering technology, PCR-mediated chromosome duplication (PCDup), was developed in Saccharomyces cerevisiae that enabled the duplication of any desired chromosomal region, resulting in a segmental aneuploid. From one round of transformation, PCDup can duplicate a single chromosomal region efficiently. However, simultaneous duplication of multiple chromosomal regions is not possible using PCDup technology, which is a serious drawback. Sequential duplication is possible, but this approach requires significantly more time and effort. Because PCDup depends upon homologous recombination, we reasoned that it might be possible to simultaneously create duplications of multiple chromosomal regions if we could increase the frequency of these events. Double-strand breaks have been shown to increase the frequency of homologous recombination around the break point. Thus, we aimed to integrate the genome editing tool CRISPR/Cas9 system, which induces double-strand breaks, with our conventional PCDup. The new method, which we named CRISPR-PCDup increased the efficiency of a single duplication by up to 30 fold. CRISPR-PCDup enabled the simultaneous duplication of long chromosomal segments (160 kb and 200 kb regions). Moreover, we were also able to increase the length of the duplicated chromosome by up to at least 400 kb, whereas conventional PCDup can duplicate up to a maximum of 300 kb. Given the enhanced efficiency of chromosomal segmental duplication and the saving in both labor and time, we propose that CRISPR-PCDup will be an invaluable technology for generating novel yeast strains with desirable traits for specific industrial applications and for investigating genome function in segmental aneuploid.

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gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing

Cited by 22 publications

References 13 publications

Development and Application of CRISPR/Cas in Microbial Biotechnology

Development and Application of CRISPR/Cas in Microbial Biotechnology

Systematic approach for assessing whether undeletable chromosomal regions in Saccharomyces cerevisiae are required for cell viability

CRISPR-PCDup: a novel approach for simultaneous segmental chromosomal duplication in Saccharomyces cerevisiae

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