A gene knock-out by destruction of a gene sequence in a metabolic pathway for phenotype study.
The filamentous fungus Aspergillus aculeatinus has a surface consisting of many spores that can fall off, fly away and turn into new fungi. These spores contain a pigment that gives the fungus its dark color. This pigment is produced by a metabolic pathway of several enzymes, called polyketide synthases. One of these enzymes in the pathway is encoded by the albA gene, so a knock-out of this gene with a destruction of the sequence should stop the production of the finished pigment. In this case, the mushroom must lose its dark color.
To investigate this, a PAM sequence and an associated 20 nucleotide long sequence were found in the albA gene for recognition with Cas9 and sgRNA. From this, sgRNA was designed, which, together with the Cas9 gene, was inserted into a plasmid designed for fungi. This plasmid was introduced into A. aculeatinus by a temporary breakdown of their cell membrane.
An overview of the trial results can be found in Figure 12. In the figure it can be clearly seen that there is a phenotypic difference between the wild-type fungus and the genetically modified mutant fungus. The wild type contains the dark pigment, while the mutant lacks the pigment with which it is white. If a closer look is taken at the spores of fungi under a microscope, it can be seen that the mutant does not produce the black pigment in the spores anymore. Sequencing of the DNA sequences for the area that Cas9 hit shows that there has been a deletion of 8 base pairs in the mutant, which is tantamount to a frame-shift mutation. This must be due to a mutational NHEJ repair of the double-strand break caused by Cas9. The mutation is a loss-of-function mutation that has led to a dysfunctional enzyme, which has stopped the production of dark pigment. This implementation of CRISPR/Cas9 in A. aculeatinus was carried out at DTU Bioengineering, Technical University of Denmark.
Figure 12. A gene knock-out in Aspergillus aculeatinus to study the expression of the albA gene, which expresses an enzyme involved in a metabolic pathway that produces dark pigment. The wild type indicates the normal fungus, while the mutant has been subjected to gene modification with Cas9. Note the color change that has resulted from the destruction of the DNA sequence of the albA gene. Sequencing reveals that it is a deletion of 8 base pairs.
A gene knock-in by replacing a sequence that alters protein function and forms resistant rice plants.
Figure 13. Rice seedlings sprayed with herbicide. To the left is the resistant genetically modified rice plant, and to the right is the wild-type rice plant.
Source: “Engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase”, Y. Sun, X. Zhang, C. Wu, Y. He, Y. Ma, H. Hou, X. Guo, W. Du, Y. Zhao, L. Xia, Molecular Plant, 9(4):628-631 (2016).
Plants produce an enzyme called acetolactate synthase (ALS) that catalyzes the first step in the production of the essential amino acids valine, leucine, and isoleucine, which are used to build other proteins. By inhibiting the enzyme, you can stop production, which will kill the plant. This can be done with a variety of weed removers that bind to the enzyme and prevent its function. If you spread these weed removers over a rice field, you will kill all weeds, but also the rice plants. Thus, it would be very useful to make rice plants that were resistant to the weed remover.
A Chinese research team succeeded in creating genetically modified resistant rice plants, using CRISPR/Cas9 and HDR to make a gene knock-in in the ALS gene by replacing part of the sequence. The rice plants were fed a plasmid containing the Cas9 gene, a DNA template, and two pieces of sgRNA. The two pieces of sgRNA were used to carve out a sequence in the middle of the ALS gene, and this DNA sequence was replaced with the DNA template by HDR. This DNA template was almost identical to the sequence of the wild type, except that it contained some specific mutations. These mutations meant that the acetolactate synthase produced was not susceptible to binding by the weed remover, thus preventing an inhibition of the production of the essential amino acids. Thus, it is a gain-of-function mutation due to a gene knock-in.
This genetic modification resulted in viable resistant rice plants, but fault mutations were also observed due to inappropriate repair of NHEJ. Using the rice plants that were modified correctly, one would be able to plant a rice field of resistant plants. In this rice field, you will be able to remove all weeds with the weed remover, but let the rice plants thrive. In Figure 13, the experimental results can be seen, where it is clear that only the genetically modified plant thrives despite the presence of weed remover.
