GENOME EDITING

I. Definitions
A. Modern Biotechnology
- Application of in vitro nucleic acid techniques, including recombinant DNA
technology or direct injection of nucleic acid into cells or organelles
- Gene editing tools like CRISPR-CAS 9, TALENs, Zn Finger
Nucleases
- Fusion of cells beyond the taxonomic family that overcome natural
physiological reproductive or recombination barriers and that are not
techniques used in traditional breeding and selection
B. Waves of Biotechnology
- First wave: agronomic traits (biotic or abiotic stresses)
- Second wave: quality traits (improved nutrition, quality traits)
- Third wave: factories (industrials, pharmaceuticals)
- Fourth wave: renewable resources
- Biofuels from cellulosic materials
- Fifth wave: use of gene editing tools
C. Gene Editing Tools
1. Gene
2. Target organism with new gene incorporated in its genome
3. New trait expressed
- Random insertion
- Other parts of vector may be incorporated in target genome
- New: Genome editing, gene editing tools
- Zinc finger nucleases (ZFN)
- TALENs (transcription activator-like effector nucleases
- CRISPR-CAS
- Site of action is specific
- Can result in removal of single base or several bases or
mutation to knock out gene or repair gene
- Can also insert a new gene for a new or improved trait
- Other parts of vector may be removed or not incorporated
in genome
- Enables editing parts of the genome by REMOVING,
ADDING, OR ALTERING SPECIFIC sections of the DNA
sequence
- Simplest, most versatile method of genetic manipulation
D. 2020 Nobel Prize in Chemistry
- Recombinant DNA technology
- Emmanuelle Charpentier and Jennifer Doudna
- Discovered CRISPR/Cas9 “genetic scissors” used to cut DNA
- Charpentier found the key tracrRNA molecule that bacteria use to
cut and disable viruses
 - Collaborated with RNA expert Doudna to eventually reprogram the
scissors to cut any DNA molecule at a specific point making the
gene editing method viable
1. CRISPR- copying from nature
- Very old defense mechanism in bacteria
- In the 1980s, scientists observed a strange pattern of clusters of DNA
repeats in bacteria, with unique sequences in between the repeats
(clustered regularly interspaced short palindromic repeats)
- The unique sequences turned out to be bits of viruses that infect the
bacteria which can recognize and defend against those viruses next time
they attack
2. CAS
- Set of enzymes called Cas (CRISPR-associated proteins) can precisely
remove the DNA out of the invading viruses
- The genes for the Cas are always near the CRISPR sequences
- CRISPR is like a wanted gallery of invading viruses
3. CRISPR and Cas
- Microbe can make the CRISPR-Cas gun down any invading virus
- The spacer DNA sequences in the gallery are copied into RNA molecules
- These RNA molecules then bind with the Cas enzymes and together drift
through the cell
- If they meet virus genetic material that matches and pairs with the
CRISPR RNA the Cas enzyme will then chop the DNA in two, preventing
the virus from duplicating
4. Components of commercial genome editing tools:
- Cas9: an endonucleasae that induces a double-strand break in genomic
DNA
- Guide RNA (gRNA): This RNA has a scaffold sequence which is needed
for Cas9 binding and a 20-nucleotide user-defined part to target DNA
sequence
- Homologous Recombination (HR) template (optional): a piece of DNA that
you would like to introduce flanked by regions of homology
- The editing process is completed by repairing the break using the
endogenous Non-homologous End Joining (NHEJ) pathway
5. Delivery systems
- Cas9 plasmid, gRNA, and HR donor vector
- Agrobacterium tumefaciens delivery
- Particle bombardment
- Lipid transfection
- Electroporation
- Microinjection
- Lipid or Liposome-mediated gene delivery
- Use of cationic lipids e.g. DOTMA-NNN-trimethylammonium
chloride
 - Cationic (+) lipids better than neutral, why? Easy, highly
reproducible, efficient
- Electroporation
- Physical transfection method
- Uses electrical pulse to create temporary pores in cell membranes
- Through which substances like nucleic acids can pass
- Electric potential is applied which drives DNA across the
membrane thru the pores
- Electric field strength and length of time of exposure are varied to
optimize
- Direct DNA transfer–naked DNA
- Physical injection of DNA directly into the nucleus of cultured cells
- Technically difficult and laborious (ok with large cells)
- Integration of foreign DNA occurs very low frequency but can be
expressed for several generations
- Efficiency of integration varies in diff organisms
- Has been applied in gene therapy, gene editing etc.
II. Understanding gene editing principles
III. Step by step procedure in gene editing
IV. Applications:
A. Health
- CRISPR gene editing cells with edited genes injected into a person with
aggressive lung cancer
- To treat sickle cells, single base mutation; tried in mice with some success
- Stanford uses CRISPR to cell
- Chinese scientists to pioneer first human trial (lung cancer)
- A team led by Lu You, an oncologist at Sichuan Univ West China
Hospital in Chengdu, July 2016
- Lu’s team will extract immune cells called T cells from the blood of
enrolled patients
- Using CRISPR tech, the gene for PD-I normally acts as a check
on cell’s capacity to launch an immune response, to prevent it
from attacking healthy cells
- Gene edited cells will be multiplied
- Reintroduced into patient’s bloodstream
- Engineered cells will hopefully act on cancer cells
- US trial is similar but in addition to knocking out PD-I gene, it will also KO
a second gene and insert a third gene before cells are reintroduced in the
patient
- Patient has Hunter’s syndrome
- Was injected with gene editing tool (CRISPR-CAS which cut out
the defective promoter of the albumin and replaced with a
powerful working promoter)
 - Jennifer Doudna and Melanie Ott reported that with a single guide RNA
they could detect as few as 100,000 viruses per micromiliter of solution
adding a second guide RNA, they detected as few as 100 viruses per
microliter
B. Agriculture
- Anti browning white button mushrooms (Agaricus bisporus)
- CRISPR knock out of one of six PPO genes reducing the
enzyme’s activity
- Waxy corn
- Less amylopectin
- FAD2KO soybean

APPLICATIONS: PLANT BIOTECHNOLOGY

1. Tissue Culture
- Sterile in vitro cultivation of plant parts such as organs, embryos, and seeds as
well as single cells on either solidified or liquid nutrient medium
- Art of plant tissue culture needs mastery of techniques. The culturist must know
how to best remove an immature embryo from a single seed without damaging its
regenerative ability
- Discerning which are good materials (calli) to move forward
- Science component:
- Culture room
- Laminar flow hood or “clean cabinet” provides clean air to avoid
microbial contamination
- Inside it is an alcohol lamp, jar of ethyl alcohol for flame
sterilization
- Aseptic conditions
- IPB Plant Tissue Culture: orchids, fruit crops such as mango, banana, avocado,
garlic, shallots, abaca, calamansi, coconut, and ornamental crops

A. Basic principles
1. Totipotency
- Each cell is an independent unit which possesses a genetic
information capable of generating into a complete organism
- Allows the plant and animal to maintain its genetic makeup or its
potential, thus regenerants will have the same genetic makeup as
the parent materials
2. Competency
- Endogenous potential of a given cell or tissue to become a fully
formed organ
- E.g. a cell or group of cells destined to become an embryo,
shoot, or root
3. Plasticity
 - Plants (and animals) are able to adapt to changes in the
environment through alteration of metabolic processes involved in
growth and development
B. Applications of Plant Tissue Culture
1. Micropropagation
- Rapid and practical micropropagation of planting materials that are
robust, of high quality, and disease-free
- Seedlings or plantlet from germinating seeds vs tissue cultured
plant materials
2. Production of important biochemicals using cell culture technologies
- Catharanthus roseus (Madagascar periwinkle). This plant
produces two major alkaloids: vincristine and vinblastine used in
cancer chemotherapy
3. General scheme for biochemicals production by plant cell cultures
- Wide cultivated plants → high producing plants → primary callus
→ stable high-producing strains → high producing strains in
optimal condition → mass culture in bioreactor
4. Somoclonal variation
- Mutation that arises from the continuous subculture of plant
tissues prior to regeneration
- IPB developed tomato somaclonal variants that are salt tolerant
5. Physical Mutagenesis
- Example: bananas galore btv-resistant vs btv-infected (bunchytop
virus resistant lakatan- mutation breeding through irradiation of
shoot tips)
- Finoforce magmutate ang chemicals para magdevelop ang
resistance o yung trait na gusto natin
6. Chemical mutagenesis
7. Genetic engineering
- Plant tissue culture plays a critical role in plant genetic engineering
- Direct organogenesis: piliin ang genetic interest at yun ang
pararamihin
8. Developmental Phases in Transgenic Plant Technology
- Gene discovery (plant tissue culture/laboratory) → transformation
→ greenhouse → field trial → commercialization
- Simplified protocol for papaya transformation:
Solo papaya → zygotic embryos → somatic embryos → squashed
somatic embryos → particle inflow gun
C. Key Information
- PLC application:
- In vitro propagation technique for asexually and sexually propagated
plants
- Production of important biochemicals in health and industry
- As a technique to generate genetic variation in plants
 - As an important component in transgenic plant technology

2. Molecular Markers
A. DNA Markers for plant characterization
- DNA: has entry and exit tolls as markers, a DNA strand contains many genres
and to find our gene of interest we need markers to locate it
B. DNA markers
- Specific dna fragments that can be identified within the whole genome
- Molecular marker:
- Any site/locus in the genome of an organism at which the dna
base sequence varies among different individuals of a population
- Such markers generally have no apparent effect on the phenotype
of the individual, but they can be determined by biochemical
analysis of DNA and are used for chromosome mapping, dna
fingerprinting, and genetic screening
- Any unique dna sequence that can be identified within the whole
genome
- Can be used to identify individuals, track genetic relationships,
identify specific traits
C. DNA markers in plant breeding
- Could identify sources of desirable genes in the germplasm
- Identify offsprings that carry the desirable genes at early stages of
development: single seed analysis or at seedling stage
- Tag desirable varieties
- Aid in settling ownership of variety in cases of infringement (assign IP for
variety protection)
- Quality control - ascertain purity of seed lot
D. DNA fingerprinting
- Basic principle: every organism has a characteristic phenotype or physical
appearance due to a unique hereditary composition
- Thus, every individual has a unique DNA fingerprint in a like manner,
humans have unique fingerprints
- DNA fingerprinting is the characterization of one or more relatively rare
features of an organism’s genome or hereditary makeup
- In DNA fingerprinting, it is very important to search for molecular markers
that can distinguish one individual from another
- In foreign markets, a red blush in mango increases its aesthetic value and
therefore its consumer preference, hence a team of scientists at IPB
discovering the DNA marker for the red blush and breeding carabao
mangoes with the trait, thick peeled mangoes are also used to improve
shelf life and post harvest qualities
E. Types of DNA Markers
- Early developed: RAPD, RFLP, ALP, VNTRS
- More robust: SSR, SNP, gene specific markers
Molecular Marker Description

Gene sequence-specific amplification Marker derived from the conserved

polymorphism (GSSAP) sequences of gene interest

Single Nucleotide Polymorphism (SNPs) Single base mutations spread along the
individual’s genome and cause the
expression of a different trait from that
exhibited by the wild type

Simple Sequence Repeats (SSR) Short nucleotide sequences that are repeated
in tandem in the individual’s genome

1. SSR
- Short sequences of nucleotides (2-6 units in length) that are repeated in tandem
- Example:
- di-nucleotide repeat: CACACACA
- Tri-nucleotide repeat: ATGATGATGATG
- It is believed that errors occur during DNA replication and extra sets of these
repeated sequences are added to the strand. Eventually, these repeated
sequences differ in length between one cultivar and another
- DNA fingerprinting analysis in Papaya: 6 genes
- Barcoding: short nucleotide sequence from a strand of genetic locus for use in
species identification
- Some applications of DNA barcodes:
- Taxonomy: species identification
- Genetic diversity analysis
- Consumer protection: identification of adulterants in processed
food products (meat, fish)
- Forensics- identification of illegally traded wildlife

3. Crop improvement by modern biotechnology

- Pinoy GM crops:
- Golden Rice: PhilRice/IRRI
- Bt Eggplant: IPB/UPLB
- Delayed ripening papaya: IPB/UPLB
- Foreign developed crops
- GM Corn: YieldGard, Bt Corn, IR Corn
1. Resistant to the Asiatic corn borer
2. Bacterial gene Bt Cry1ab was transferred to corn by genetic engineering
- GM Corn: RR Corn (NK 603)
1. Resistant to the herbicide called glyphosate
2. Bacterial gene CP4 epsps was transferred to corn by genetic engineering
 - Bt Cotton
1. Bt cotton is an insect resistant transgenic crop designed to withstand the
cotton bollworm therefore the crop requires little pesticide
2. Philippine Fiber Industry Development Authority is planning to use Bt
Cotton to help revive the country’s cotton industry
- Plant Biopharming
- Production of pharmaceutical or industrial proteins in plants
- Use of plants as “green factories” for useful proteins in health and industry
- Advantages of plants vs bacteria/mammalian cell culture
- Investments in infrastructure and cost of production are significantly
smaller in plant systems
- New gene editing techniques:
- Applications in agriculture:
- Anti-browning white mushrooms
- Penn state university (2016)
- Reduced formation of melanin, improving shelf life of the
mushroom
- CRISPR knock-out of one of six PPO genes, reducing the
enzyme’s activity
- Waxy corn (Zea mays)
- DuPont Pioneer
- Altered starch composition
- CRISPR-deletion of waxy gene Wx1
- No plant pests or genetic material from plant pests were
used in developing product
- Corn is not listed as federal noxious weed
- Therefore, APHIS does not consider CRISPR-Cas waxy
corn to be regulated pursuant to 7CFR part 340
- FAD2KO soybean
- High oleic acid, low linoleic acid soybean oil
- TALEND-directed deletion within the FAD2-1A and
FAD2-1B genes (enzymes for oleic acid and linoleic acid
synthesis
- Delivery thru Agrobacterium
- Resulted in inactivation of the two genes

Overview of Genetic Engineering

1. Isolation of gene
- Extraction of DNA
- Cloning of gene
2. Modify gene and prepare gene construct
- Incorporated in vector
3. Transformation
- Agrobacterium
 - Particle Bombardment: Gene gun
- Tissue culture: Indirect organogenesis (callus)
4. Regeneration
5. Improvement of transgenic plant (conventional breeding)
- Greenhouse selection/testing
- Field testing
- Variety