CRISPR‐Enabled Tools for Engineering Microbial Genomes and Phenotypes

Tarasava, Katia; Oh, Eun‐Suok; Eckert, Carrie A.; Gill, Ryan T.

doi:10.1002/biot.201700586

Cited by 35 publications

(24 citation statements)

References 201 publications

(272 reference statements)

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“…The pioneering development 13 of a mycobacterial knockdown system based on CRISPR interference (CRISPRi) ( Figure 1A) presents an efficient alternative for large-scale library construction, with the capacity for rapid validation and the potential to titrate repression 14 . However, since CRISPRi depends on the use of custom single guide RNAs (sgRNAs) which can have variable and unpredictable efficacies, and given the paucity of guidelines for the design of sgRNAs for variant Cas9 endonucleases 15 , initial characterization of the ability of a specific guide sequence to repress transcription represents a key requirement for the subsequent use of CRISPRi in phenotypic assays.…”

Section: Introductionmentioning

confidence: 99%

CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

Wet

Gobe

Mhlanga

et al. 2018

Preprint

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High-throughput essentiality screens have enabled genome-wide assessments of the genetic requirements for growth and survival of a variety of bacteria in different experimental models. The reliance in many of these studies on transposon (Tn)-based gene inactivation has, however, limited the ability to probe essential gene function or design targeted screens. We interrogated the potential of targeted, large-scale, pooled CRISPR interference (CRISPRi)-based screens to extend conventional Tn approaches in mycobacteria through the capacity for positionally regulable gene repression. Here, we report the utility of the "CRISPRi-Seq" method for targeted, pooled essentiality screening, confirming strong overlap with TnSeq datasets. In addition, we exploit this high-throughput approach to provide insight into CRISPRi functionality. By interrogating polar effects and combining image-based phenotyping with CRISPRimediated depletion of selected essential genes, we demonstrate that CRISPRi-Seq can functionally validate Transcriptional Units within operons. Together, these observations suggest the utility of CRISPRiSeq to provide insights into (myco)bacterial gene regulation and expression on a genome-wide scale.

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Section: Introductionmentioning

confidence: 99%

CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

Wet

Gobe

Mhlanga

et al. 2018

Preprint

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“…In addition, in order to enable efficient regulation of multiple pathways simultaneously, we attempt to integrate the endogenous QS element with the gene repression CRISPRi tool (EQCi) for pathway-independent regulation. CRISPRi, which is developed based on the DNase inactivated CRISPR/Cas endonucleases, such as 5 / 39 dCas9 (20,21), has been emerged as effective tool to fine-tune the expression of multiple target genes (22,23). Importantly, using CRISPRi, gene repression strength could be optimized by adjusting the expression of dCas9 and sgRNA, or by changing the targeting position of sgRNA (21,24).…”

Section: Introductionmentioning

confidence: 99%

Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in industrialStreptomyces

Tian

Yang

et al. 2020

Preprint

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Dynamic regulation has emerged as an effective strategy to improve product titers by balancing metabolic networks, which can be implemented by coupling gene expression to pathway-independent regulatory elements, such as quorum-sensing (QS) systems.However, these QS-based circuits are often created on heterologous systems and must be carefully tuned through tedious testing and optimization process to make them work well, which hampers their application in industrial microbes including streptomycetes.In this study, we design a pathway-independent QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycinproducing strain Streptomyces rapamycinicus. EQCi has the advantages of both the QS system and CRISPRi, which enables tunable, fully autonomous and dynamic regulation of multiple targets simultaneously. To demonstrate its effectiveness, we downregulate three key nodes in essential pathways separately to divert metabolic flux toward rapamycin biosynthesis. In each case, significant increases in rapamycin titers are achieved. We further apply EQCi to simultaneously control these three key nodes with proper repression strength by changing sgRNA targeting positions. The final rapamycin titer reaches to 1836±191 mg/L, which is the highest reported titer. Notably, compared to traditional static engineering strategy, which result in growth arrest and suboptimal rapamycin titers, EQCi regulation substantially promote rapamycin titers without affecting cell growth, which indicates that it can achieve the trade-off between essential pathways and product synthesis. Collectively, this study provides a simple and effective 3 / 39 strategy for optimizing product titers and may have the potential to apply to other industrial microorganisms.

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“…While the CRISPR/Cas9 technology is now utilized extensively for engineering microbial genomes [19] and editing targeted nucleotide without DSBs, [20] examples remain rare for its application in basidiomycetous yeasts, except the pathogenic yeast Cryptococcus neoformans. [21][22][23][24] Here, we report successful construction of a CRISPR/Cas9 system for genetic manipulation of R. toruloides.…”

Section: Introductionmentioning

confidence: 99%

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides

Jiao

Zhang

Liu

et al. 2019

Biotechnology Journal

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The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. Here, the development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon‐optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single‐guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI, CAR2, and CLYBL gene, respectively, transforming into those Cas9‐expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI, gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome‐editing tool in R. toruloides, which should facilitate functional genomic study and advanced cell factory development.

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CRISPR‐Enabled Tools for Engineering Microbial Genomes and Phenotypes

Cited by 35 publications

References 201 publications

CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in industrialStreptomyces

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides

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