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Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity.

ABSTRACT: Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by more than 100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.

SUBMITTER: Jiang W

PROVIDER: S-EPMC7169367 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity.

Jiang Wenyan W Oikonomou Panos P Tavazoie Saeed S

Cell 20200227 5

Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gen ...[more]

PMID: 32109417

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Project description:Goss's bacterial wilt and leaf blight is a disease of maize caused by the gram positive bacterium Clavibacter michiganensis subsp. nebraskensis (Cmn). First discovered in Nebraska, Goss's wilt has now spread to major maize growing states in the United States and three provinces in Canada. Previous studies conducted using elite maize inbred lines and their hybrids have shown that resistance to Goss's wilt is a quantitative trait. The objective of this study was to further our understanding of the genetic basis of resistance to Goss's wilt by using a combined approach of genome-wide association mapping and gene co-expression network analysis. Genome-wide association analysis was accomplished using a diversity panel consisting of 555 maize inbred lines and a set of 450 recombinant inbred lines (RILs) from three bi-parental mapping populations, providing the most comprehensive screening of Goss's wilt resistance to date. Three SNPs in the diversity panel and 10 SNPs in the combined dataset, including the diversity panel and RILs, were found to be significantly associated with Goss's wilt resistance. Each significant SNP explained 1-5% of the phenotypic variation for Goss's wilt (total of 8-11%). To augment the results of genome-wide association mapping and help identify candidate genes, a time course RNA sequencing experiment was conducted using resistant (N551) and susceptible (B14A) maize inbred lines. Gene co-expression network analysis of this time course experiment identified one module of 141 correlated genes that showed differential regulation in response to Cmn inoculations in both resistant and susceptible lines. SNPs inside and flanking these genes explained 13.3% of the phenotypic variation. Among 1,000 random samples of genes, only 8% of samples explained more phenotypic variance for Goss's wilt resistance than those implicated by the co-expression network analysis. While a statistically significant enrichment was not observed (P < 0.05), these results suggest a possible role for these genes in quantitative resistance at the field level and warrant more research on combining gene co-expression network analysis with quantitative genetic analyses to dissect complex disease resistance traits. The results of the GWAS and co-expression analysis both support the complex nature of resistance to this important disease of maize.

Dataset Information

Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity.

Publications

Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity.

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