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The J. Craig Venter Institute’s mission is to be a “...not-for-profit genomic research institute dedicated to advances in human health and the environment.” At JCVI, The Minimal Genome Project began in 1995 when the mycoplasma genitalium genome was sequenced, and an ethical review was published in 1999. The first bacterial genome, and the first self-replicating synthetic cell, containing 523 genes, was completed in 2010, leading up to the first minimal cell publication in 2016. The minimal cell was then systematically tested to find which genes for which were essential for the cell’s survival and growth and removed those that were not. This produced the Mycoplasma mycoides JCVI-syn3.0 minigenome with only 473 genes. This is by far the smallest semi artificially designed organism. Analysis of these essential genes is ongoing and the next logical progression of this project.1
Problem Statement
Problem Statement
About one third of the original genes remain unknown. In order to test the function of these genes without killing the cell, CRISPR interference (CRISPRi) has been chosen. CRISPRi allows for sequence-specific gene expression control by blocking transcription of specific regions of DNA by binding a catalytically inactive Cas9 protein bound to a customizable single guide RNA (or sgRNA) that matches the target gene.2 This highly customizable aspect of the sgRNA makes it so the complexes could target essentially any location in the genome. The Cas9-sgRNA complex then binds to the complementary target DNA region, creating a steric block, which prevents elongation during transcription by RNA polymerase, resulting in the target gene being repressed. This alteration does not affect the underlying DNA structure like a knockout would, so the induction or repression of genes is reversible.
Objectives
Objectives
There is currently no well studied model that allows further insight into the mechanism of genes essential to life; therefore, a simplified cell with a genome wide CRISPRi library is needed in order to better uncover gene functionality.
First, we aim to use the relationship between genotype and phenotypic changes to evaluate gene functions thus uncovering some of the currently unknown basic biological processes.1 By understanding gene function, genes that are truly essential to a living organism can be established. Establishing an accurate minimal set of genes will fuel discovery in the fields of medicine and biology.
The goal of this project is to create a successful minimal cell CRISPRi gene library which should be made up of minimal cell strains that characterize all 473 essential genes in the minimal cell.The ability to produce our engineered plasmids in E.coli and grow with successful CRISPR inactivated genes after Mycoplasma transformation, allows the genes to be studied. It is also crucial that all the genes be characterized by different gRNA sequences and DNA barcodes with no mutations in the backbone.
Our final goal is to publish findings of a completed library with mutations. Sharing these discoveries allows other scientists to benefit from our work. It also validates the amount of time, research, and effort that has been invested and opens up the project for new applications and collaborators.
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