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Design Overview
We are deciphering the function of minimal cell genes by reducing the expression of the genes one-by-one with CRISPRi and deducing the function of the gene based on the effects the perturbation produces. Our strategy involves 1) constructing the sgRNA-dCas9 plasmid for each gene, 2) performing CRISPRi by transforming the minimal cells with the plasmid, and 3) sequencing the transformed minimal cells and analyzing the effects of CRISPRi.
Plasmid Construction
Plasmid Construction
The sgRNA-dCas9 plasmid for each gene is constructed through a series of 3 PCR reactions and E.coli transformation.
PCR 1: It incorporates a random 8bp barcode into the sgRNA fragment corresponding to a gene of the minimal cell, assigning a unique signature to the fragment.
PCR 2: It adds a spacer complementary to the target gene of the minimal cell into the sgRNA-barcode fragment.
PCR 3: It amplifies the sgRNA-barcode fragment that will be used to assemble the sgRNA-dCas9 plasmid.
E.coli Transformation: It assembles the complete plasmid consisting of sgRNA, barcode, and inducible dCas9 plasmid by using the E.coli's machinery and recombination.
Quality Control: Upon extracting the plasmid using DNA extraction, the identity of the plasmids are confirmed by amplifying the sgRNA-barcode region through PCR and 1) visualizing its size through gel electrophoresis and 2) sequencing the region.
CRISPR Interference
CRISPR Interference
After plasmids are built, transformation is performed by introducing the sgRNA-dCas9 plasmid into the minimal cells via polyethylene glycol (PEG) mediated DNA uptake. The transformed cells are then grown and selected on an antibiotic containing agar plate. Colonies selected from the plate are incubated in liquid mycoplasma media. A fraction of the culture is used for quality control and the rest is freeze stored as a part of the CRISPRi library. For quality control, the sgRNA-barcode fragment is PCR amplified. The identity of incorporated fragment is then confirmed through 1) gel electrophoresis and 2) Sanger sequencing.
Sequencing Analysis
Sequencing Analysis
Sequencing data of the transformed cells is used to identify the minimal cell gene corresponding to the sgRNA by identifying the incorporated sgRNA and comparing its sequence to the sgRNA-gene database. The CRISPRi library is completed for the gene when two or more minimal cell strains with unique barcodes are established.
CRISPRi library construction is still ongoing for a number of genes. Once the library is completed, the design will move towards understanding the function of the genes. The library will be treated with tetracycline in order to induce the activation of dCas9 to decrease the expression of the minimal cell genes. These cells will be sequenced to perform functional genomics and transcriptomics/proteomics analysis to understand the effects of each gene's knockdown from a molecular level.
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