How It Works
** Make sure to understand what DNA, RNA, protein, gene expression, and the Central Dogma theory are before reading this page. For now, it is sufficient to just know CRISPR as a gene editing tool, as you might have heard before.**
Our various CRISPRkit versions have different objectives, but they all aim to demonstrate how gene expression is altered by CRISPR. In essence, the main goal of all our experiments is as follows:
Overview:
CRISPRkit involves three main components essential for conducting a successful experiment. In this section, we will introduce you to each of these components and explain their roles in our CRISPR experiments.
Chromoproteins - The target protein, or more specifically, the protein that contains the gene we are trying to use CRISPR on (to clarify, we're not actually editing/cutting the gene. Instead, we're using CRISPR to modulate and alter gene expression by repressing it without creating breaks in the DNA) To ensure that the results of the CRISPR experiment are easily visible, the choice of protein is crucial. We chose to use a chromoprotein-based reporter system for this purpose.
Chromoproteins are conjugated proteins that contain a pigmented cofactor. One common example is hemoglobin, which contains a heme cofactor that makes blood appear red. In our experiment, chromoproteins are selected as the reporter system. While fluorescence proteins are more commonly used in laboratory settings and suitable for techniques such as flow cytometry to track and visualize biological processes, they only emit color under a specific wavelength of light. In contrast, chromoproteins can produce bright colors that are visible under an external light source, making them ideal for low-tech biology experiments. We can see vivid colors and marvel at CRISPR’s remarkable ability to regulate gene expression with our naked eyes.
The chromoproteins come in the form of plasmids, which are double-stranded circular DNA.
Image of red chromoprotein (circular shape)
However, plasmids cannot express on their own. As you can see, chromoproteins come in the form of a transparent liquid. How can they express color? Traditionally, plasmids are introduced into bacteria and undergo protein synthesis in a process called bacterial transformation. However, this involves complicated steps such as plating and growing the bacteria under stringent conditions. One central task of ours was to simplify these steps.
Plasmids, surprisingly, do not need to be incorporated into bacteria to achieve expression. Instead, we can stimulate expression of chromoproteins using a cell-free transcription-translation (TXTL) system. This innovative approach streamlines the process and significantly reduces the steps required for plasmid expression.
The TXTL (translation-transcription) system - The buffer solution that enables the expression of DNA/RNA/protein to occur in a cell-free context. It creates a microenvironment where all the necessary components are mixed in, allowing the process of central dogma and our experiment to take place.
Transcription-translation (TXTL) is a powerful technique that enables cell-free protein expression in a test tube. To create a cell-free extract, bacterial cells are lysed, and the resulting solution is centrifuged to remove debris such as membranes and organelles. This leaves behind an extract that contains the necessary transcription and translation machinery for protein expression. Additional components such as amino acids, energy sources, and cofactors are added to the extract to support protein production.
In our experiments, we will mix the chromoproteins and guide RNA (gRNA) plasmids, as well as the dCas9 protein into the TXTL buffer. The plasmids undergo transcription and translation through the central dogma transcription-translation process, while the dCas9 protein is already in its final form and does not require additional processing. (see below for description of CRISPR components: gRNA & dCas9) The effectiveness of CRISPR is evaluated by visualizing the resulting protein expression in the tubes. TXTL is an essential and versatile component that plays a critical role in simplifying the steps required for gene editing and making our kits highly accessible and user-friendly.
Image of cell free extract process
Image of adding plasmid / dcas9 to tubes for crispr
CRISPR-dCas9 system - The heart of our experiment which enables precise modulation of our reporter system’s gene expression (chromoproteins). By using this system, we can achieve gene repression without creating any breaks in the DNA.
The CRISPR-dCas9 system is a crucial part of our kit. As mentioned above, you may have heard of CRISPR as a gene editing tool, specifically CRISPR-Cas9. This system finds the target gene and induces a double-stranded break (DSB), thus destroying its ability to express and produce functional proteins. However, we are using CRISPR-dCas9 instead for our kits. This system finds the target gene using the same mechanism, but it does not induce DSB. Instead, it simply binds to the DNA without altering its structure, enabling the modulation of gene expression levels without permanently damaging the target gene. (Learn more about CRISPR-Cas9 & CRISPR-dCas9 in the Concepts page)
CRISPR-dCas9 has two components to it
a. gRNA (Learn more here)
The gRNA
b. dCas9 (Learn more here)
Either recruit or block