Construction of a CRISPR-I Library to Characterize Gene Function in a Minimal Cell

Overview

A minimal bacterial cell, JCVI-syn3.0, derived from Mycoplasma mycoides has been constructed at the J. Craig Venter Institute. It operates with only 473 genes, of which 149 have unknown function. The manipulation of gene activity in the minimal cell is expected to enable the characterization of these genes by tying genotype to phenotypic changes, thus, uncovering some of the currently unknown basic biological processes. For this purpose, a variation of the CRISPR-Cas9 system, CRISPRi, will be systematically implemented for each gene in the genome such that the gene expression can be repressed. In a series of three PCR reactions each followed by E. coli transformation, a plasmid composed of a gene-specific gRNA, a random DNA barcode, and a tetracycline-inducible gene for dCas9, a nuclease-defective Cas9 protein used in CRISPRi, was assembled for each of the genes in the JCVI-syn3.0 genome. Subsequently, the plasmids were transformed into the minimal cell using a Cre-lox recombination system. The current workflow was established using three target genes as test cases in which various methods to assemble the final plasmids were examined. The plasmids assembled for the three selected target genes were successfully generated and introduced into the mycoplasma genome. We have begun the scaled-up procedure and presently, we have assembled plasmids with site-specific gRNAs for about 360 genes. The approach involving PCR and E. coli transformation is effective for making a genome-scale CRISPRi library and the same workflow will be followed for the remaining genes until we have a complete library. We will then examine the phenotypic effects from the repression of gene expression. Each gene will be individually silenced and a whole-genome transcriptome map will be constructed using RNA-sequencing. A comparison between gene expression in CRISPRi strains and wild-type strains can be drawn from the map, indicating up-regulated and down-regulated genes. CRISPRi-targeted genes with similar expression profiles will then be clustered in a heat map, revealing information about its role and how it affects the expression of other genes. The complete characterization of genes in JCVI-syn3.0 is expected to elucidate a set of functions common to all life forms.

Competition history

• AJAS 2020 Category not listed

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