Dataset Information

High-resolution characterization of gene function using single-cell CRISPR tiling screen.

ABSTRACT: Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Here, we present "sc-Tiling," which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response.

SUBMITTER: Yang L

PROVIDER: S-EPMC8249386 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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High-resolution characterization of gene function using single-cell CRISPR tiling screen.

Yang Lu L Chan Anthony K N AKN Miyashita Kazuya K Delaney Christopher D CD Wang Xi X Li Hongzhi H Pokharel Sheela Pangeni SP Li Sandra S Li Mingli M Xu Xiaobao X Lu Wei W Liu Qiao Q Mattson Nicole N Chen Kevin Yining KY Wang Jinhui J Yuan Yate-Ching YC Horne David D Rosen Steven T ST Soto-Feliciano Yadira Y Feng Zhaohui Z Hoshii Takayuki T Xiao Gang G Müschen Markus M Chen Jianjun J Armstrong Scott A SA Chen Chun-Wei CW

Nature communications 20210701 1

Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing ...[more]

PMID: 34210975

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Project description:Salmonella enterica is represented by >2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials. S. enterica is the leading bacterial cause of foodborne illness in the United States, with well-established detection methodology. Current surveillance protocols rely on the characterization of a few colonies to represent an entire sample; thus, minority serovars remain undetected. Salmonella contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer contents of these can be considered serovar specific. We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq (serotyping by sequencing of the CRISPR loci), to identify multiple serovars present in a single sample. Using mixed genomic DNA from two Salmonella serovars, we were able to confidently detect a serovar that constituted 0.01% of the sample. Poultry is a major reservoir of Salmonella spp., including serovars that are frequently associated with human illness, as well as those that are not. Numerous studies have examined the prevalence and diversity of Salmonella spp. in poultry, though these studies were limited to culture-based approaches and therefore only identified abundant serovars. CRISPR-SeroSeq was used to investigate samples from broiler houses and a processing facility. Ninety-one percent of samples harbored multiple serovars, and there was one sample in which four different serovars were detected. In another sample, reads for the minority serovar comprised 0.003% of the total number of Salmonella spacer reads. The most abundant serovars identified were Salmonella enterica serovars Montevideo, Kentucky, Enteritidis, and Typhimurium. CRISPR-SeroSeq also differentiated between multiple strains of some serovars. This high resolution of serovar populations has the potential to be utilized as a powerful tool in the surveillance of Salmonella species.IMPORTANCESalmonella enterica is the leading bacterial cause of foodborne illness in the United States and is represented by over 2,600 distinct serovars. Some of these serovars are pathogenic in humans, while others are not. Current surveillance for this pathogen is limited by the detection of only the most abundant serovars, due to the culture-based approaches that are used. Thus, pathogenic serovars that are present in a minority remain undetected. By exploiting serovar-specific differences in the CRISPR arrays of Salmonella spp., we have developed a high-throughput sequencing tool to be able to identify multiple serovars in a single sample and tested this in multiple poultry samples. This novel approach allows differences in the dynamics of individual Salmonella serovars to be measured and can have a significant impact on understanding the ecology of this pathogen with respect to zoonotic risk and public health.

Dataset Information

High-resolution characterization of gene function using single-cell CRISPR tiling screen.

Publications

High-resolution characterization of gene function using single-cell CRISPR tiling screen.

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