CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression in<i>Saccharomyces cerevisiae</i>

Zhai, Haotian; Cui, Li; Xiong, Zuhong; Qi, Qingsheng; Hou, Jin

doi:10.1093/nar/gkac463

Cited by 11 publications

(8 citation statements)

References 49 publications

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“…A few transcription factors had been cloned from the yeast genome and fused to the C‐terminus of dCas12a as the new AD candidate. Here we tested HAP4 (Stebbins & Triezenberg, 2004) and MED2, which is from the tail part of the mediator (Jeronimo et al, 2016; Petrenko et al, 2016) and has been confirmed to improve transcription (Zhai et al, 2022). The commonly used activator VPR was also included as a positive control.…”

Section: Resultsmentioning

confidence: 99%

Nanopore sequencing improves construction of customized CRISPR‐based gene activation libraries

Wang,

Tan,

Lian

et al. 2024

Biotech & Bioengineering

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Clustered regularly interspaced short palindromic repeats (CRISPR)‐based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in‐house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase‐mediated non‐amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a‐based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing‐based workflow demonstrated here should provide a cost‐effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.

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Section: Resultsmentioning

confidence: 99%

Nanopore sequencing improves construction of customized CRISPR‐based gene activation libraries

Wang,

Tan,

Lian

et al. 2024

Biotech & Bioengineering

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“…In addition, a synchronized gene inhibition and activation system has been developed, providing more flexible and powerful tools for the metabolic engineering of S. cerevisiae. 104…”

Section: Crispri/crispra Systems Based On Dcas9 or Dcas9 Fused With T...mentioning

confidence: 99%

“…Increasing the recruitment of transcriptional effectors in the promoter region may improve the likelihood of achieving higher levels of transcriptional inhibition or activation . In addition to the RNA scaffold-based methods mentioned above, the strategies of recruiting inhibition/activation domains through protein scaffolds have also been developed. , For example, the protein scaffold-mediated signal amplification systems, SPY and SunTag, have been constructed in S. cerevisiae . In addition, a synchronized gene inhibition and activation system has been developed, providing more flexible and powerful tools for the metabolic engineering of S. cerevisiae …”

Section: Crispr/cas9-based Systems Used For Regulation Of Gene Transc...mentioning

confidence: 99%

“…In addition to the RNA scaffold-based methods mentioned above, the strategies of recruiting inhibition/activation domains through protein scaffolds have also been developed. , For example, the protein scaffold-mediated signal amplification systems, SPY and SunTag, have been constructed in S. cerevisiae . In addition, a synchronized gene inhibition and activation system has been developed, providing more flexible and powerful tools for the metabolic engineering of S. cerevisiae …”

Section: Crispr/cas9-based Systems Used For Regulation Of Gene Transc...mentioning

confidence: 99%

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Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools

Liang,

Gao,

et al. 2024

ACS Synth. Biol.

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The CRISPR/Cas9 systems have been developed as tools for genetic engineering and metabolic engineering in various organisms. In this review, various aspects of CRISPR/Cas9 in Saccharomyces cerevisiae, from basic principles to practical applications, have been summarized. First, a comprehensive review has been conducted on the history of CRISPR/Cas9, successful cases of gene disruptions, and efficiencies of multiple DNA fragment insertions. Such advanced systems have accelerated the development of microbial engineering by reducing time and labor, and have enhanced the understanding of molecular genetics. Furthermore, the research progress of the CRISPR/Cas9-based systems in the production of high-value-added chemicals and the improvement of stress tolerance in S. cerevisiae have been summarized, which should have an important reference value for genetic and synthetic biology studies based on S. cerevisiae.

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“…A CRISPR-mediated regulator recruiting system has been developed to amplify transcriptional activation and repression. This system has been developed by fusing protein scaffolds (SPY and Sun Tag systems) with nuclease-deficient CRISPR proteins so that several effectors are recruited for accurate and efficient multi-regulation of target gene transcription ( Zhai et al, 2022 ).…”

Section: Recent Technologies and Future Trendsmentioning

confidence: 99%

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

Wu,

Feng,

Sun

et al. 2023

Front. Bioeng. Biotechnol.

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Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

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CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression inSaccharomyces cerevisiae

Cited by 11 publications

References 49 publications

Nanopore sequencing improves construction of customized CRISPR‐based gene activation libraries

Nanopore sequencing improves construction of customized CRISPR‐based gene activation libraries

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

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