Gen knock-in and knock-out

This subpage constitutes the second part of the theory for Biotech Academy’s material on CRISPR-Cas9.

The molecular changes in the DNA sequences of genes have a direct impact on the function of the genes. You can both remove gene functionality, by gene knock-outs, and add new features to a gene, by gene knock-ins. When working with gene modification, one is interested in how changes in the genotype express themselves in the phenotype of the organism. These changes to the organism can be very violent, or almost invisible, depending on the function of the gene.

Gen knock-out

Genes code for proteins that have a specific function. If you destroy the gene, you prevent the production of the protein, with which you will not observe that particular function. Mutational interventions can cause loss-of-function mutations. A loss-of-function mutation causes a knock-out of a gene, which is characterized by the gene producing dysfunctional protein or not producing enough protein. Thus, the function of the protein is reduced or removed. With gene knock-outs, it is thus possible to prevent specific functions and phenotypic traits by influencing the function of the executive proteins. You can optionally make a knock-out with Cas9 by destruction or excision. Knock-outs can also be performed with an insertion in the middle of a gene sequence or by replacing some of the sequence.

By making knock-out mutants and comparing them with wild types (non-modified organisms), where the gene is still functional, you can study a gene and its associated protein functions in a given organism.

An example of the use of gene knock-outs is the alteration of an organism’s metabolism. Often, the cell’s molecules are produced by a wide range of processes, called metabolic pathways. Each step of the process is associated with specific enzymes that catalyze the modification at each step. Enzymes are proteins and therefore encoded by genes, with which it is possible to make knock-outs of them. A knock-out in a gene encoding an enzyme in the metabolic pathway will mean that the process will stop at the step catalyzed by the enzyme due to the lack of functional enzyme. It could be that one was interested in obtaining a substance that was not the product of the metabolic pathway, but merely an intermediate step. In this way, it was possible to make a knock-out in the gene for the enzyme that further converts the desired substance, with which the substance will accumulate at that particular step, as it cannot be further metabolized in the metabolic pathway.

Gen knock-in

A gene knock-in is the insertion of a new DNA sequence at a very specific position in the DNA. This DNA sequence can be inserted in extension with the DNA that already holds or replace a DNA sequence. A knock-in can cause a gain-of-function mutation, where the insertion of a DNA sequence alters functions or adds functions to the protein that the gene codes for. You can also enhance functions by increasing the expression of the gene, which gives you more protein. By knocking in entire coding sequences, you can introduce new proteins and thus new functions to the organism. Knock-ins can be performed with Cas9, by making an insertion or a replacement of a DNA sequence.

Knock-ins have been widely used to create disease models that study the effect of genes on a disease. Knock-ins can also insert reporter genes in extension with other genes that you want to investigate. Reporter genes produce a protein that can easily be seen has been produced. Since the reporter gene is linked to another gene’s transcription, you can see how much protein is produced from the studied gene. A classic example of a reporter is green fluorescent protein (GFP), which is a green-lit protein. By associating the gene for GFP with another gene by a knock-in, you can see how much the studied gene is expressed and where in the organism the protein is, based on observation of the luminous protein.