Gene Editing Techniques & Applications PDF

I. CRISPR CAS9 - Clustered Regularly Interspaced Palindromic Repeats and crispr-associated protein 9 - a site-specific gene editing technology. - It is a component of bacterial immune systems that can cut a target DNA then shutting the targeted gene off. - Edits...

I. CRISPR CAS9 - Clustered Regularly Interspaced Palindromic Repeats and crispr-associated protein 9 - a site-specific gene editing technology. - It is a component of bacterial immune systems that can cut a target DNA then shutting the targeted gene off. - Edits genes and harnesses natural DNA repair processes to modify gene/s in the desired manner - Consists of : 1. Cas 9 - protein/nuclease that can cleave through DNA - Endonuclease that causes double-stranded sequence to break, allowing modifications to the genome 2. Guide RNA/ sgRNA - can recognize the sequence of DNA to be edited - Matches with the desired gene HISTORY 1987- Atsuo Nakata & his team of researchers from Osaka University, Japan first reported the presence of Crispr in E coli/ Escherichia Coli Mid 2000s - functionality & importance of the Crispr Cas 9 system was recognized in prokaryotes. Crispr system is a part of prokaryotic adaptive immunity, which protects prokaryotes from viral DNA attack from bacteriophages & plasmids Adaptive Immunity- immunity of an organism acquired after exposure to an antigen or vaccination DEVELOPERS OF CRISPR CAS9 Emmanuelle Charpentier - Microbiologist Jennifer Doudna - Biochemist They were the 1st to propose that the bacterial Crispr cas 9 system could be used as a programmable toolkit for genome editing in humans and other animal species PURPOSE 1. Edit genes 2. Research 3. Treat genetic disorders 4. Agriculture 5. Medicine Development PROCESS A. How Crispr Cas9 works in prokaryotes B. How Crispr Cas9 is applied in Genetic Engineering Pros & Cons of Crispr Cas9  Pros Immunotherapies Cons Cure Genetic Diseases Germlining Drug development Bioweapons Improve crops Reduced Genetic diversity Safety risk I.FLAVR SAVR TOMATO - The first genetically engineered food. - Developed by Calgene, Inc. Raymond Carlyle Valentine May 21 1994 - sometime on 1997 - Alternate to Commercial based Tomato - Made by silencing the RNA responsible for the production the production of pectin (component of tomatoes’ wall) PROCESS A. HOW FLAVR SAVR TOMATO IS MADE 1. Duplicate & Isolate the PG gene from a normal tomato, synthesizing the Antisense PG gene 2. Antisense PG cDNA will silence the PG protein enzyme 3. Insertion of Antisense Pg cDNA into plant cell B. HOW IT IS INSERTED INTO PLANT CELL(Antisense PG cDNA -> Agrobacterium Tumifaciens/infector) 1. Antisense PG cDNA will be cut by Restriction enzyme type 2 into pieces 2. Antisense PG cDNA will be inserted in a bacteria’s plasmid (multiply), needs CaMV- promoter that controls the binding of RNA polymerase to DNA 3. E Coli acts as host of the plasmid (fastest & easiest cultivator under a monitored area) 4. E Coli carries plasmid -> (transfer by triparental mating) Agrobacterium tumefaciens (Can infect plant cells & transfer a defined sequence of their DNA to the plant cell). Agrobacterium tumefaciens is renamed to ti-plasmid 5. Ti-plasmid is introduced to tomato/ GENE GUN II.BT CORN - Bt corn, short for Bacillus thuringiensis corn, is a genetically modified type of corn that has been engineered to produce insecticidal proteins from bacteria. - Discovered by Shigetane Ishiwata & Ernst Berliner - provides resistance to pests by incorporating genes specific to corn pests that produce insecticidal proteins from Bacillus thuringiensis - targets specific pests such as lepidopteran larvae (corn borers) and coleopterans (corn rootworm beetles). - PROCESS 1. Bacillius thiurengensis DNA is completely isolated in a laboratory. 2. In the lab, the particular DNA sequence that makes up the Cry1Ab gene is discovered and replicated. 3. The Cry1Ab soil bacteria form of the gene has been slightly changed to work better in maize plants. 4. In a laboratory, a maize plant gets transformed with the novel modified Cry1Ab gene, which provides insecticidal protection. 5. The corn plant lacking ideal genes for field production undergoes the final stage in biotechnology, where plant breeders cross it with their best-performing lines to produce a high-yielding BT corn variety. - PURPOSE: Reduced Dependency on Chemical Insecticides Improved Pest Management  Cost- Effective Solution Environmental Benefits III.AQUADVANTAGE - AquaAdvantage salmon is a genetically engineered Atlantic salmon designed to grow faster than conventional salmon, reaching market size in about half the time. This was achieved by adding a growth hormone gene from the Chinook salmon to an Atlantic salmon. APPLICATION: Genetic engineering is applied to fertilized Atlantic salmon eggs to create AquaAdvantage salmon. This process involves inserting a specific gene construct into the egg's DNA. PROCESS 1. Atlantic Salmon/Target Body for preferred meat 2. Chinook Salmon/Growth Hormone 3. Ocean Pout/Promoter 1. To avoid violating natural laws, AquaAdvantage salmon is kept indoors and not let out in the wild, to avoid causing a disruption in the ecosystem.  2. From the produced offspring, the female eggs are pressure shocked to ensure they are infertile/sterile to avoid mass population. 3. Only female offspring are sold, remaining male offspring are kept in storage for the production of more salmon. It is not encouraged for an AquaAdvantage salmon to mate with other organisms, which makes the creation of this process an exclusive practice in the industry. IV.GENE DELIVERY - Gene delivery is introducing foreign genetic material, such as DNA or RNA, into host cells. This technique is used in various areas of biotechnology, medicine, and research, particularly in gene therapy, where the goal is to treat or prevent disease by correcting or modifying genes within a patient’s cells. Gene delivery must reach the host cell's genome to induce gene expression. Viral Methods - Viral vectors are modified viruses that can efficiently deliver genes into cells, removing viral DNA, and using the virus as a vehicle to deliver the therapeutic DNA. Viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. (Integrating = exhibit constant expression of the defective gene product). (Main drawbacks: immunogenicity & cytotoxicity). The most common Viral Vectors include: 1. Adeno Virus - DNA viruses able to infect a wide range of cells and deliver large amounts of genetic material - Ideal for transient gene expression/ rapid high-level gene delivery is needed -APPLIED IN: Gene therapy, vaccine development (ex. COVID-19 Vaccines), research 2. Lentivirus - Subclass of retroviruses, that can infect dividing & non-dividing cells and can integrate into the host genome for stable expression - Best for long term gene expression - APPLICATION: study gene function and regulation - Employed in the development of gene therapies for genetic disorders, creating stable cell lines for research, modifying stem cells for therapeutic applications 3. Retrovirus - RNA viruses that integrate their material into the host cell’s genome, typically infecting only dividing cells - Idea for stable gene transfer & long term expression in dividing cells - APPLIED IN:Cancer research, create stable cell lines for research - Cancer gene therapies,engineering cells for research, creating models for studying gene Non-Viral Methods  - Non-viral Methods are generally considered safer and less immunogenic than Viral Methods. Easier to target cells/tissue, to manipulate. Is biocompatible, tissue specific, very flexible, cost effective. Unfortunately less efficient than Viral Vectors/methods. Prone to In vivo toxicity (lipid components). Physical Vectors 1. Gene Gun - Or biolistics, is a method that uses a high pressure device to shoot microscopic particles with DNA into target cells - Ideal for transferring cells/tissues that are difficult or other methods (plant/animal tissues) - APPLICATION: Plant engineering & research - Creates GMOs , vaccines and research 2. Electroporation - Uses electric pulses (current to break cell wall) to create temporary pores in the cell membrane (specifically, the phospholipid bilayer), allowing DNA to enter. However, its strength varies according to the tissue it encounters. - Uses an electroporator - Cuvette - removable container from electroporator, contains the therapeutic DNA/plasmid  3. Microinjection - Precise technique involving a fine needle to directly inject genetic material into individual cells - For specificity/precision, such as fertilized eggs, embryos single cells in research - Used in creating transgenic animals, gene editing, etc. - Used in GMO, ex. Transgenic mice - Can only penetrate matter at cellular level Chemical Vectors - Similar functions, identical & shell-like, differing composition - All applicable in gene therapy 1. Polymeric - natural/Synthetic polymers that can condense DNA into nanoparticles, facilitating cell entry. Can be natural (cellulose, chitin, starch) or synthetic (PLGA/Poly(lactic acid-co-glycolic acid), PLA/Poly(lactic acid), PGA/Poly(glycolic acid)), and they are used for nanoparticle generation/production. Present in Research/Biotechnology applications. Polymeric nanoparticles (PNPs) are the most common materials studied as nanocarriers for drugs and gene delivery. 2. Liposomes - Spherical vesicles made of lipid bilayers that can encapsulate DNA/RNA/drugs merging with cell membrane to deliver contents - Vaccine development  3. Peptide - Polypeptide chain = amino acid only - Short amino acid sequence designed to bind to genetic material, and assist in crossing cell membranes - - Ideal for speedy & specific teams - Experiment, cancer studies, research V.HARDY WEINBERG EQUATION - By Godfrey H. Hardy, mathematician & Wilhelm Weinberg, Physician - States that a population's allele & genotype frequencies are constant, UNLESS there is some type of revolutionary force acting upon them. - Despite some minor differences in appearance, a species’ allele & genotype frequencies remain constant - EXAMPLE: frogz >_< - GG- dominant allele - Gg- Heterozygous genotype - gg- Recessive allele ASSUMPTIONS OF HARDY WEINBERG EQUILIBRIUM 1. No selection (no physical advantages) 2. No mutation 3. No migration 4. Large Population 5. Random Mating *Not completely applicable in real life, however it makes a good support/baseline on how an evolving population is changing/remains constant ALLELE FREQUENCIES p+q=1  GENOTYPE FREQUENCIES p^2 + 2pq + q^2 = 1 VI.EVOLUTION & ORIGIN OF BIODIVERSITY:Patterns of Descent with Modification I.Reproductive Isolating Mechanisms (What keeps species distinct) A. Pre-zygotic Barrier/ Isolation Mechanisms -Barriers present before mating 1. Geographical/Ecological/Habitat Isolation - Potential Mates occupy different areas or habitats, thus, they never come in contact - A geographical entity separates the two species, inherently leading to isolation 2. Temporal or Seasonal Isolation - Different groups may not be productively mature at the same season/month/year - Implies different mating seasons for differing species/ different mating seasons 3. Behavioral Isolation - Courtship pattern varies between each species group - ex. Singing, dancing, cleaning (birds) 4. Mechanical Isolation - Differing reproductive organs, prevents successful inbreeding - Physical attributes of varying species’ reproductive organs are incompatible 5.Gametic Isolation - Incompatibilities between egg & sperm prevent fertilization; either physical or chemical barrier prevents fertilization - Ex.sea urchin species simultaneously shed their gametes but remain evolutionarily distinct - Species A Egg = Species B sperm B. Post-Zygotic Barrier/Isolation Mechanisms -barriers after mating process -common issues are within offspring called hybrid 1. Hybrid Inviability - fertilized eggs fail to develop past embryonic stages - Mating occurs, but no pregnancy occurs 2. Hybrid Sterility - Hybrids are sterile because gonads develop abnormally  - Abnormal separation of chromosomes - Gene cannot be passed down further 3. Hybrid Breakdown - Offspring of hybrid is sterile; gene cannot be passed down further - F1 hybrids are normal, vigorous and viable, but F2 contains many weak or sterile individuals - II.Modes of Speciation (How Speciation Happens) 1. Allopatric Speciation - Change of environment introduces new species - occurs when biological populations become geographically isolated from each other to an extent that prevents or interferes with gene flow 2. Sympatric Speciation - evolution of a new species from a surviving ancestral species while both continue to inhabit the same geographic region - Genetic mutation occurs - Occurs when there is no geographical isolation but rather behavioral isolations 3. Parapatric - occurs when a smaller population is isolated, usually at the periphery of a larger group, and becomes differentiated to the point of becoming a new species. - Occurs when a smaller group of the main population is geographically isolated. - Occurs when one species is spread out over a large area. VIII. Pedigree Analysis Gene - DNA sections that code for specific proteins/functional RNA, playing a crucial part in biological functions Allele - refers to the variation of genes, leading to diverse traits such as eye color, hair type DNA/deoxyribonucleic acid - Molecule that carries genetic information in living organisms - Resembles a twisted ladder (Double Helix) and is found inside the nucleus of our cells Punnett Square Genotype - unique sequence of DNA Phenotype - observable expression of this genotype - a person's presentation. *Dominant allele masks the effect of the Recessive allele *A recessive allele only appears if there are 2 copies possessed GENETICS Gregor Mendel - Father of Genetics - 1st important studies of heredity - Identified specific traits in the garden pea and studied them through generations MENDEL’S CONCLUSIONS 1. Law of Segregation - 2 alleles for each trait separate when gametes from; parents only pass 1 allele for each trait to each offspring 2. Law of Independent Assortment - Genes for different traits are inherited independently of each other Ex. F1 Green x F1 Green = F2 3/4 Green & ¼ yellow AUTOSOMAL VS. SEX-LINKED Autosomes - All chromosomes except sex chromosome Sex chromosomes - 23rd; X & Y DOMINANT VS. RECESSIVE Dominant - Masks the other trait; the trait that shows IF PRESENT - Represented by capital letters Recessive - Trait that can only be expressed when a dominant allele is NOT PRESENT - Will only show if both alleles are present - Represented by lowercase letters MODE OF INHERITANCE Dominant A. Autosomal - Huntington’s Disease (brain disorder that causes mental illness) - Dwarfism - Polydactyly (extra fingers or toes) B. Sex-linked - Vitamin D-resistant rickets Recessive A. Autosomal - Albinism(no pigmentation)  - Cystic Fibrosis(excessive mucus production in lungs) - Sickle cell anemia (red blood cells are shaped like a “C” instead of round) B. Sex-linked - Color Blindness - Hemophilia DOMINANT (affected individuals have RECESSIVE affected parents) Autosomal X-Linked Autosomal X-Linked A. Affected A. Mother-son A affects both A. Mostly males don’t B. Father-all genders (1:1 ratio) affects males have an daughters (Overwhelmi affected / ng number) mother B. Affects both C. father-son B. mother-son / genders (1:1 x ratio) TERMS Genotype - Genetic makeup of an organism Phenotype - physical characteristics of an organism Dominant allele - Phenotypically expressed over another allele Recessive Allele - Expressed in absence of a dominant allele Autosomal trait - Trait located on an autosome (non–sex chromosome) Sex-linked Trait - Trait located on one of the two sex chromosomes Homozygous - Having 2 identical alleles for a particular gene Heterozygous - Having 2 different alleles for a particular gene