Introduction
The following biological report discusses the human manipulation techniques relating to transgenesis and selective breeding of cows. Transgenesis refers to the removal of beta-lactoglobulin protein from milk produced by cows to reduce immune reactions due to allergies to the protein. This is done through multiple techniques; this report refers to CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-Cas9) and zinc finger nucleus mRNA. Selective breeding refers to breeding for cows to produce a2 milk to reduce stomach discomfort and aggravation of diseases (Jianqin, S., Leiming, X., Lu, X., Yelland, G., Ni, J., & Clarke, A. ‘Effects of milk containing only A2 beta casein versus milk containing both A1 and A2 beta casein proteins on gastrointestinal physiology, symptoms of discomfort, and cognitive behaviour of people with self-reported intolerance to traditional cows’ milk’, 2019). Multiple techniques are used for selective breeding in cows; this report focuses on marker assisted selection and artificial insemination. I have decided to select cows as the case studies as it directly affects New Zealand as dairy farming is currently our main contributor to the GDP (gross domestic product) and may provide insight to the processes that are used to produce milk for consumers and biological implications that could be a result of these processes.
Biological Concepts (Transgenesis)
Transgenesis is experimental biology involving exogenous gene insertion into the genome. This process is followed by a germ-line transmission (reproduction process of one male and one female forming a zygote through deriving embryo stem cells of a blastocyst from transgenesis) and analysis of the offspring. This process is usually used to alter the characteristics of an exogenous gene.
For the removal of beta lactoglobulin from milk produced by cows is done by gene targeting, which comes under transgenesis. Gene targeting is the deletion of an endogenous gene to affect the germ-line or phenotype of the organism. Usually this process is used in experimentation, however, it has been used to create BLG (beta lactoglobulin) free milk. This process has been done through multiple techniques, this report focuses on CRISPR-Cas9 and zinc finger nuclease.
Beta lactoglobulin (BLG) is a compact, folded globular protein. It usually linked to an identical molecule (dimer) making it pressure sensitive. There are different variations of the protein, the most common being A. It exists as a dimer between 5.2 and 7 pH and as a monomer at 3 or above 8 pH. Its denaturing process links to its function as it produces a gel-like substance. This is used mostly in whey protein. Beta lactoglobulin compromises of eight antiparallel beta strands (strands connecting latterly to two or three H-bonds), with a triple turn alpha-helix. This is at the end of a conical barrel. Beta lactoglobulin is found in cows’ milk and has no equivalent protein in humans. The protein can create strong allergic reactions, resulting in large levels of antibodies generated. Removing the protein is deemed the safest way to reduce these allergies and provide nutrition to a wider market (Rands, ‘The use of gene editing in the primary industries’, 2019)
Crispr-Cas9 is a gene editing tool that has the ability to change DNA. The name CRISPR refers to the DNA sequences found in bacteria and other microorganisms (short and partially palindromic repeated). CRISPR in these organisms plays a large part in their immune system, destroying the genome of invading viruses.
The image to the right shows how CRISPR works in bacteria (CRISPR-mediated immunity). The DNA sequence consists of short repeats (diamonds) and spacers (squares).
When a new virus is detected, a spacer is created to exist among other spacers as genetic memory. This sequence is processed to form CRISPR RNA molecules. These molecules guide ‘molecular machinery’ to a matching DNA sequence in the virus, triggering the ‘machinery’ to cut the genome.
Humans can now manipulate this process for gene editing through insertion and deletion (knock in and knock out) to make accurate and precise edits in DNA. CRISPR contains four genes that allow this to occur: cas9, tracrRNA (trans-activating), pre-crRNA and sgRNA (single guide). Pre-crRNA fuses to tracrRNA which allows cas9 to be directed to very specific section of DNA that matches the sequence of crRNA. The cas9 then makes the changes necessary as a single transcript (sgRNA). The sgRNA guides the cas9 binding protein to the DNA targeted site. SgRNA then signals Cas9 to cut DNA where needed and make edits given the sgRNA. CRISPR the uses a protospacer adjacent motif (PAM) to prevent the Cas9 being targeted as it may recognise itself as a virus (Petersen, ‘Basics of genome editing technology and its application in livestock species’, 2019).
Zinc finger nucleases (ZFN) are artificial restriction enzymes that are created by joining DNA-binding zinc fingers with endonuclease Fok 1 (DNA cleavage domain). This creates a very specific genome cutting tool ZFN has been used as a gene targeting tool, changing the genome through mutagenesis (change in genetic information through mutation by nature or mutagens). The structure of a zinc finger consist of 30 amino acids forming two beta sheets (a form of secondary structure of a protein) which are opposite an alpha helix. ZFN causes a site specific double strand break in DNA. This induces a response by the organism to go through the natural DNA-repair process, usually homologous and non-homologous recombination (homologues is when two similar or identical molecules of DNA switch nucleotide sequences, nonhomologous is when the ends of the breaks are ligated together). Both repair processes are able to be manipulated to make genetic modifications to the DNA. For homologous recombination, a model DNA template is made In a lab for the natural repair process to use. Nucleotides are then attached to match the model DNA, therefore changing the genome from the original. Nonhomologous recombination is used to delete genes within DNA as no model DNA is used to repair, the ends are joined through ligation.
Biological Implications (Transgenesis)
An implication found when using CRISPR is ‘off-target’ genetic changes. This is when Cas9 protein, which uses single guide RNA, has been unintendedly directed to cleave DNA sections which do not match the single guide RNA code. This seems to be at high frequency, around 50 percent (Zhang, Tee, Wang, Huang & Yang, ‘Off-target Effects of CRISPR/Cas9-mediated Genome Engineering’, 2019). This may result in mutations that could affect the phenotype of the offspring and has caused debate of germline editing. The effects include ‘large deletions or mutations’ that occur thousands of base pairs from the site where Cas9 was supposed to cut (Dockrill, ‘BREAKING: Crispr Could Be Causing Extensive Mutations and Genetic Damage After All’, 2019). For cows, this could cause potentially, serious problems for the dairy industry. A misguided Cas9 that goes undetected could cause a defective gene or lethal genes where death is the likely outcome. Cas9 could be accidently guided to cut A2 caseins instead of beta lactoglobulin, rendering the milk close to having no value. This mutation could spread throughout the dairy industry causing a massive problem for humans and possibly for cows who now are unable to function properly due to missing switches, genes or the creation of defective genes due to misguided Cas9 from sgRNA. CRISPR has also been proposed to change the diversity of cattle. The removal of a protein may spread throughout the industry. This means that the allele for beta lactoglobulin will no longer exist, essentially reducing biodiversity. Long term effects are unknown as this is a recent technology, but it is likely that beta lactoglobulin may not exist in milk due to evolution from the selective pressure of CRISPR as it is eliminated from the genome.
Zinc finger nuclease has been found to have implications where it has caused improper formations of proteins and overexpression of genes. This is usually due to ZFN specification being low causing random integration of donor DNA (DNA that has been ‘designed’ as the new DNA to be inserted in intended area) and/or multiple random cuts (in the hundreds) in the genome. ZFN causes these problems mainly when studying the localisation of proteins using green fluorescent tag (GFP). The protein created has greater number of folds than the endogenous levels have specified resulting in an over expressed gene. As a result, protein misfolding occurs affecting the protein function from greater levels of gene expression. The issue may even affect interactions between proteins and produce dominant effects from different alleles. This may lead to chromosomal changes and rearrangements or even cellular death. This creates implications for the dairy industry in which, beta-lactoglobulin may not be the only thing cut from the genome. This would cause milk to change from its normal structure as proteins are cut out of the genome (the milk may not be milk anymore). If this goes undetected, it will cause the dairy industry to collapse as the form of milk changes due to the genome of milk changing. The removal of multiple proteins from milk could cause reading frame shifts in the genome or result in the natural repair process become prone to error as it is overwhelmed with breaks. This could lead to mutations in the milk resulting in changes in the evolution as the genome is passed on if undetected.
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