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CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis , a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria.
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Advances in CRISPR-enabled genome-wide screens in yeast
Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome-wide screens are powerful tools for unraveling genotype–phenotype relationships, enabling precise manipulation of genes to study and engineer industrially useful traits. Traditional genetic methods, such as random mutagenesis or RNA interference, often lack the specificity and scalability required for large-scale functional genomic screens. CRISPR systems overcome these limitations by offering precision gene targeting and manipulation, allowing for high-throughput investigations into gene function and interactions. Recent work has shown that CRISPR genome editing is widely adaptable to several yeast species, many of which have natural traits suited for industrial biotechnology. In this review, we discuss recent advances in yeast functional genomics, emphasizing advancements made with CRISPR tools. We discuss how the development and optimization of CRISPR genome-wide screens have enabled a host-first approach to metabolic engineering, which takes advantage of the natural traits of nonconventional yeast—fast growth rates, high stress tolerance, and novel metabolism—to create new production hosts. Lastly, we discuss future directions, including automation and biosensor-driven screens, to enhance high-throughput CRISPR-enabled yeast engineering.
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- Award ID(s):
- 1922642
- PAR ID:
- 10582632
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- FEMS Yeast Research
- Volume:
- 25
- ISSN:
- 1567-1364
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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