Genetics 344 - SU3 – Genome Editing

LECTURE 1: INTRODUCTION TO GENOME EDITING

PRE-GENOME ENGINEERING ERA

GENETIC ENGINEERING

 The modification of an organism’s genome sequence by inserting, deleting or

changing individual nucleotides or nucleotide sequences (potentially entire
genes or several genes simultaneously).
 The broad genome engineering process (horizontal gene transfer) happens in
nature (eg. HIV and agrobacterium).
 Requires two crucial steps: a double strand break (DSB) mechanism and a DSB
repair mechanism.

A BRIEF HISTORY OF GENOME ENGINEERING

1st generation (genetic modification)

 based on fundamental recombinant DNA technology (GM) that is present in

nature.
 Ie. plasmid vectors, restriction enzymes, DNA ligases, bacterial transformation,
genetic transformation of organisms (microbes, animals and plants).
2nd generation (genome editing)

 Using programmed nucleases (GE)

 Meganucleases, zinc finger nucleases
(ZFNs), transcription activator-like e ector
nucleases (TALENs), CRISPR/Cas9

GENETIC TRANSFORMATION OF MICROBES

 Human insulin expression in [Link]

(Humulin R) was the first commercial
biotech product (1982) 

GENETIC TRANSFORMATION OF ANIMALS

 DNA is injected directly into the nucleus of the fertilized egg

 Endocytosis and the use of liposomes (artificially created vesicles) can be used
in the delivery of genetic material into animal cells. These fuse with the
membrane and release their contents into the cell using a process called
conjugation.
 YACs (a type of plasmid vector), when used in conjunction with microinjection,
allow for the introduction of large pieces of genetic material into animal cells.
 Retroviruses (such as HIV)
 Examples of genetically transformed animals include AquaAdvantage salmon
(grow bigger than normal salmon), GloFish, Enviropig (produces less phosphate
in their faeces).

GENE THERAPY

 a technique that uses a gene(s) to

treat, prevent or cure a disease or
medical disorder
 Has been used for disorders such as
severe combined immunodeficiency
(SCID) and a mutation in the
adenosine deaminase (ADA) gene.
GENETIC TRANSFORMATION OF PLANTS

 Agrobacterium tumefaciens
o able to transfer a portion of its DNA (specifically T-DNA) into the genomes
of plant cells
o Ti-plasmid: The tumor-inducing plasmid found in Agrobacterium
tumefaciens. It contains genes responsible for virulence and tumor
formation.
o T-DNA (Transfer DNA): A specific segment of the Ti-plasmid that gets
transferred into the plant cell during infection. Scientists can manipulate
the T-DNA by inserting genes of interest.

 Direct DNA transfer (biolistic plant transformation) - Coating microscopic gold or

tungsten particles with DNA and shooting them into plant tissues.
 Mutation breeding is a traditional technique where plants are exposed to
chemicals or radiation to induce mutations. These mutations can lead to
desirable traits (e.g., increased yield, disease resistance). Unlike genetic
engineering it depends on random changes occurring within the genome.
 pCAMBIA vectors are a series of plant transformation vectors that are widely
used in genetic engineering to introduce foreign DNA into plant cells

Transgenics - Refers to organisms that have been

genetically modified to contain genes from other
species (transgenes)

Cisgenics - transferring genes from a closely

related species
 Lecture 2: First generation genetic engineering (GM food)
*little from this lecture will be asked, more for context than content

WHAT IS GM FOOD

GM food, or genetically modified food, refers to food products derived from organisms
whose genetic material (DNA) has been altered using genetic engineering techniques.
This modification is done to achieve specific traits that are considered beneficial for
agriculture, nutrition, or food production. While GM foods o er numerous advantages in
agriculture and nutrition, they also raise concerns about safety, environmental impact,
and ethical considerations.

AGRICULTURAL BIOTECHNOLOGY (AgBiotech) BREEDING AIMS

 Herbicide resistance
o Roundup Ready soybean, maize and cotton (modified to resist glyphosate
herbicide, primarily through the introduction of a modified EPSPS gene
which codes for important amino acids).
 Insect resistance
o Bt (this is a gene developed from Bacillus thurigiensis. It is a toxin that
kills worms that try to eat the plant).
 Disease resistance
o Fungal and bacterial diseases
o Viruses
 Altered nutrition composition
o Golden rice
o Resveratrol (antioxidant found in the skin of grapes)
 BioPharming – the use of plants to manufacture pharmaceutical products
o High value proteins/metabolites
o Vaccines (subunits of the hepatitis B vaccine
have been produced in bananas, Gardasil an
HPV vaccine has been produced in tobacco
plants).
 Stress tolerance
o Drought
o Salinity
o Cold
SHORTCOMINGS OF GENETIC ENGINEERING

 We cannot control the insertion site(s) in the plant genome (can only be
controlled in genetic editing).
 Cannot control the insert copy number
o May lead to multiple copies of a gene which may result in gene silencing
o Agrobacterium vs direct DNA transfer: direct DNA transfer has a higher
likelihood of multiple copies
 Expression of ‘foreign’ proteins may lead to toxicity/allergenicity
 Selectable (antibiotics) and screenable (fluorescent proteins) markers
o The use of antibiotic resistance markers may inadvertently contribute to
the spread of resistance genes in microbial communities, posing a public
health risk.
o Screenable markers, such as fluorescent proteins (e.g., GFP), allow
researchers to visually identify transformed cells without selective
pressure
 Horizontal gene transfer
o Genetically modified traits could transfer to non-target species, leading to
unintended ecological impacts, such as the development of
"superweeds" that are resistant to herbicides.
 Development of resistance
o The emergence of resistant pest of weed populations can undermine the
long-term e ectiveness of genetically modified traits, leading to a cycle of
escalating pesticide use and resistance development.

CURRENT STATUS OF GM FOODS

 Currently GM foods only make up about 20% of all crops farmed worldwide
 We don’t have enough food to supply the rapidly growing population with the
current agricultural practices
 Biotech crops will allow us to meet the challenges of increased population and
climate change.
 How do biotech crops contribute to food security, sustainability and climate
change mitigation? They increase crop productivity, conserve biodiversity (save
land from plowing and cultivation), provide a better environment (decreased
herbicide and insecticide use), reduce CO2 emissions, help alleviate poverty
and hunger

Figure 1: TOP 5 BIOTECH CROPS

GMO REGULATION (NB)

The GMO act (Act 15 of 1997) + the five

di erent levels of regulation

 Laboratory and greenhouse facility

registration - facilities are
inspected for compliance with GM
guidelines and licensed for a 5-year
period.
 Field trials – field testing of GM
crops in defined and fenced fields
for multiple seasons
 Commercial releases - need to
provide safety and risk trials (in
case of allergenicity/toxicity) before
they can be released for general
use in food production
 Commodity imports – imports for
commercial use as food or feed or
processed products. No planting in
SA.
 Labelling (only partially covered by the act) – labelling of food when it has 5% or
more GM content

The act does not cover genome editing as it was not defined as a process when the act
was written. The definitions in the act were those existing at that time (in 1997).

THE ISSUES – GM FOOD DEBATE

 Despite multiple studies having been conducted on the topic (>600 safety
assessments and >2000 research studies), some people are still concerned
about the safety of GM foods.
 GM has been accused of being an inexact science (due to not being able to
control where in the genome the genes insert or the copy no. variation). Although
the same can be said of conventional breeding and mutation breeding (a
traditional plant breeding technique that involves inducing mutations in the
genetic material of plants to create new varieties with desirable traits), both of
which are considered acceptable by the general population.
 Precision breeding or ‘re-wilding’ uses modern genome editing techniques to
give traits back to plants that had long ago been bred out of them.
o We may similarly be able to use GE to bring back plants from extinction
THE FUTURE OF GM CROPS

 Each GM event has its own unique characteristics. Understanding these

di erences is essential for assessing the future of GM crops, their role in
agriculture, and their acceptance by consumers and regulators.
 Most people don’t understand genetic modification which results in fear of
GMO’s. The public needs to be educated on GM so that it can be more widely
accepted.
 GM food priorities
o Benefits aimed at consumers rather than corporate agriculture
 Genome editing!!

_________________________________________________________________________________

Lecture 3: 2nd generation GE – programmed nucleases

PROGRAMMED NUCLEASES

 Site directed nucleases (SDNs) or sequence specific nucleases (SSNs)

 These are endonucleases (enzymes that cleave the phosphodiester bond within
a polynucleotide chain) directed to specific DNA sequences through specific
DNA-binding activity
 Contain a DNA binding domain and a catalytic (DNA cutting) domain
 Introduces a double strand break (DSB) in a specific DNA sequence
 Includes the following: V1.0, V2.0
o Meganucleases (MN’s)
o Zinc Finger Nucleases (ZFNs)
o Transcription Activator Like E ector Nucleases (TALENs)
o Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR
associated protein 9 (CRISPR/Cas9)
 Nucleases cut DNA and then rely on natural systems to fix it again.
MEGANUCLEASES (aka homing nucleases)

 Endonucleases that have large recognition sites (20-40 bp)

o Site generally only occurs once in any given genome (see block one)
o Found in most organisms – Archaea, bacteria, phages, fungi, yeast, algae
and some plants

BLOCK ONE
Restriction enzyme 6 base cutter: random sequence, chance to get that
specific 6 bp recognition site?

4^6 = 4096

ie. Theoretically a 6 base cutter will cut every 4096 bases on average.

What about a 12 – 40 base cutter? 4^12 = 1.68x10^7, 4^40 = 1.2x10^24. With

the human genome 3x10^9 bp in length, even cutting once is a chance event.

 Two main groups exist:

o Intron endonucleases - enzymes that recognize specific sequences within
introns and cleave them. This cleavage is an essential step in the splicing
process, where introns are removed, and exons are joined to form mature
mRNA
o Intein endonucleases – specialized enzymes that play a role in the
process of protein splicing. They are segments of proteins that can
catalyze their own removal from a precursor protein, allowing the
surrounding sections to be joined together.
 They are divided into five families based on sequence and structure motifs – the
most important of these is LAGLIDADG
o Mostly found in the mitochondria and chloroplasts of eukaryotic
unicellular organisms
o LAGLIDADG name derived from the conserved amino acid motif that is
found in all members of this family
 Can be exploited for GEd
o I-Scel (discovered in the mitochondria of baker’s yeast Saccharomyces
cerevisiae)
o I-Crel (from the chloroplasts of green algae Chlamydomonas reinhardtii)
o I-Dmol (from the archaebacterium Desulfurococcus mobilis)
MEGANUCLEASES AS TOOLS

*Due to their rare recognition sequence to use them is expensive and time consuming
and so this technology is largely redundant.

 High specificity of meganucleases gives them a high degree of precision and

much lower cell toxicity
 Limited by the repertoire of meganucleases discovered (a few hundred)
 There are two approaches to design and make tailor-made meganucleases
o Modifying the specificity of existing nucleases by introducing a small
number of variations to the amino acid sequence of the meganuclease or
by selecting the functional proteins on variations of the natural
recognition sites.
o Creating chimeric meganucleases (fusing protein domains from di erent
meganucleases such as I-Dmol and I-Crel to create E-Drel and DmoCre).
 Commercial applications (largely redundant)
o Precision Biosciences (American biotech company) has developed a
design process called directed nuclease editor (DNE) which is capable of
creating engineered meganucleases that target and modify a user-defined
location in a genome
o Cellectis (French biotech company) have developed a collection of over
20000 protein domains from the meganucelase I-Crel
o Bayer Crop Science used DNE to incorporate a gene sequence into the
genome of cotton plants, targeting in precisely to a pre-determined site.

ZINC FINGERS NUCLEASES (ZFNs)

 Zinc fingers are one of three DNA binding domains of eukaryotic transcriptional
factors (along with helix-turn-helixes and basic leucine zippers) that recognizes a
specific DNA sequence of 3bp and binds there
 Zing finger nucleases are artificial endonucleases generated by fusing a zinc
finger DNA binding domain to the DNA cleavage domain of a type IIS RE, Fokl
o IIS RE is a type of restriction enzymes, which cut DNA at specific
sequences allowing for the creation of overhangs
o "FokI" is a specific type IIS restriction enzyme that functions as a dimer, it
requires two FokI proteins to bind to adjacent DNA sites to create a
double-strand break.
 Modular assembly by combining three separate zinc fingers that can each
recognize a 3bp DNA sequence to generate a 3-finger array that can recognize a
9bp target site.
 Dimerization allows for an 18bp recognition site (cutting frequency = 4^18)
FokI endonuclease

This is a type IIS restriction enzyme that comes from

Flavobacterium okeonokoites. It is unique when
compared to other restriction enzymes (such as type
II) as it consists of both a N-terminal DNA binding
domain and a non-specific DNA cleavage site at the
C-terminus (it cuts outside of it’s recognition site).

For e icient DNA cleavage FokI needs to form a dimer

(two FokI units working together). When these units
dimerize, they cut both DNA strands and generate
fragments with sticky ends.

APPLICATIONS/ISSUES OF ZFNs

 When used in functional genomics it typically requires the “knock-out” of a gene

of interest, where after the e ect on the target organism is studied.
o ZFNs are used to introduce mutations in specific genes, disrupting their
function.
 The first application of ZFNs to achieve double stranded breaks (DSBs) was in
the fruitfly. Since then, similar successes were achieved in nematodes,
zebrafish, Arabidopsis, rodents and crop plants.
o Cows have been modified for altered milk proteins, pigs have been
modified for less human rejection of transplant organs and human genes
for the HIV co-receptor CCR5 have been modified as a potential
treatment for HIV/AIDS
 DISADVANTAGES
o Each ZFN should only bind a specific 3bp of DNA, they are not always
perfectly specific which may cause o -target cleavage.
o The process of designing ZFNs to target a specific DNA sequence is
complex, labour intensive, and time-consuming.
o Toxicity and immunogenicity - potential side e ects or adverse biological
responses that can occur during or after genome editing
o Very expensive ($25K for a custom made ZFN)

TALENs

 Transcription activator-like e ector nucleases are artificial restriction enzymes

generated by fusing a TAL e ector DNA-binding domain to the FokI DNA cleavage
domain.
o Same idea as ZFNs but using TALEs instead of zinc finger
 TALEs are proteins secreted by the plant
pathogenic bacterium Xanthomonas during
infection. These proteins can promoter
sequences in the host plant and activate the
expression of plant genes that aid bacterial
infection.
 TALE DNA binding domain contains a repeated highly conserved 33-34 amino
acid sequence except for the 12th and 13th amino acids which are highly variable
(repeat variable di-residue) which show a strong correlation with specific
nucleotide recognition.
 Engineered TALEs can also be fused to the cleavage domain of FokI to create
TALENs (has been shown to be e ective in a variety of organisms including
Arabidopsis, human stem cells and yeast).
o Engineered TALENs can be used to create programmed DSBs, which are
repaired by NHEJ or HDR (discussed later) to create a variety of
mutational outcomes.
o Can also be used to create knock-in mutations (such as TB resistance in
cattle).
 DISADVANTAGES
o O -target cleavage (uncertainty in the specificity of TALEN binding)
o Lack of e icient delivery mechanism
o Unknown toxicity/immunogenicity
o Expensive

Lecture 4: CRISPR/Cas as a bacterial immune system

Programmed nucleases V2.0 – CRISPR/Cas
 Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR
associated protein (Cas)
 CRISPR/Cas is an adaptive immune response that evolved in bacteria and
archaea as a result of the ongoing battle between them.
 Innate vs Adaptive Immunity: CRISPR/Cas originated as an adaptive immune
system in bacteria, providing a highly specific and learned defense against viral
invaders, while restriction enzymes represent a more general, innate immune
defence mechanism.
 Apoptosis: The use of CRISPR/Cas for gene editing can trigger apoptosis if
excessive DNA damage occurs, either from on-target or o -target cuts.
THE CRISPR/CAS IMMUNE SYSTEM (NB)

 CRISPR is the name of a genomic locus that functions as an adaptive immune

system against invading phages or plasmids
 CRISPR loci range from 24-48bp and comprise of repeats which are separated by
spacers (hypervariable) of similar length (20nts for Cas9)
 The locus encodes an endonuclease (Cas) and stores short stretches (spacers)
of foreign sequence, which are transcribed into CRISPR RNAs (crRNAs) that each
contain a variable sequence from the invading DNA (protospacer) and part of the
CRISPR repeat.
o The repeats are specific for the host organism

The bacterial (adaptive) immune response occurs through 3 steps:

Acquisition/adaptation

 When bacteria are invaded by viral DNA, “memorising” CAS protein complexes of
the CRISPR/Cas immune system select short sequences (protospacers) of the
 foreign DNA and integrate them as spacer
sequences into their own chromosome.
 Type II CRIPSR systems incorporate sequences
(spacers) from invading DNA between CRISPR
repeat sequences encoded as arrays within the
bacterial host genome

CRISPR-RNA (crRNA) biogenesis

 CRISPR loci are transcribed into long pre-crRNA,

then processes into mature crRNAs

crRNA-guided interference

 When subsequent phages infect, spacers are

transcribed from the array and each crRNA
hybridises with a second RNA (tracrRNA) and the
protospacer encoded portion of the crRNA directs
the Cas9 to cleave complementary target DNA
(viral) sequences.
 Cleavage occurs only if the protospacer is flanked
by a short sequence known as a protospacer
adjacent motif (PAM)
o The repeat sequences that flank spacers in
such CRISPR arrays lack PAMs and cannot be cleaved (CRISPR won’t
cleave itself)
o Viral protospacer sequences are flanked by PAMs and thus are targets for
CRISPR/Cas9
 Cas9 comprises of nuclease RuvC and HNH which together produce DSBs and
separately can produce single-strand nicks

CRISPR/Cas9: MECHANISM

 Based on the CRISPR system of Streptococcus pyogenes, which involves a single

Cas protein (Cas9) and two RNAs (crRNA and tracrRNA)
 It is possible to combine these two RNAs into a single chimeric guide RNA
(known as gRNA or sgRNA) that can e iciently direct Cas9 activity to specific
DNA targets in vitro and in vivo.
 The gRNA has a region of 20 nucleotides at its 5’ end, which binds the target DNA
and determines specificity. Any 20-nucleotide sequence can be placed at that
site.
  Target recognition also depends on additional interactions between Cas9 and
the target DNA, which require the presence of PAM immediately downstream of
the 20-nucleotide sequence targeted by the gRNA.
o S. pyogenes CRISPR/Cas9 requires a PAM that is NGG (know this!)
 The 3’ region of the gRNA is an invariable sequence that is required to form a
complex with Cas9
 Once a target is bound, two separate nuclease domains of Cas9 (RuvC and
HNH) are involved in cleaving each strand of the target DNA.
 Cleavage occurs within the guide RNA target region, usually three nucleotides
upstream of the PAM

Video on CRISPR/Cas9: [Link]

DNA DOUBLE STRAND BREAK REPAIR

There are two di erent pathways to repair

DSBs that operate in nearly all cell types:

1. Nonhomologous end-joining (NHEJ)

This is the predominant repair mechanism

even though it is error prone. It leads to the
introduction of insertion/deletion mutations
(indels) of various lengths which may result in the disruption of gene function (knock-
out phenotypes). It may also disrupt the binding sites of trans-acting factors in DNA
regulatory sequences (promotors) – resulting in changes in gene expression.

2. Homology-directed repair (HDR)

Homology directed repair can introduce specific point mutations into the genome. It
can also insert desired sequences through recombination of the target locus with
exogenously supplied DNA ‘donor templates which is known as a knock-in mutation.

CRISPR/Cas 9: DELIVERY AND TARGET RANGE

Functional CRISPR systems require the delivery of Cas9 as well as guide RNA.

 Cas9 may be expressed from a helper plasmid carrying the coding sequence
(fused with a nuclear localization signal) under the control of an appropriate
promoter
 Can be proved in the form of in vitro transcribed capped mRNA
 Can be delivered as a purified recombinant protein
  In some model organisms, transgenic strains have been generated that express
Cas9 in the germ line cells.

Delivery of the gRNA (guide RNA) is more complicated. In vitro transcription of gRNA is
usually carried out using the RNA polymerase of bacteriophages. Another approach is
to express the gRNA from a plasmid or transgene in vivo.

LECTURE 5: CRISPR/Cas II

Cas9 is an example of a programmable nuclease – they can be steered to a specific

spot to execute their function.

Doudna & Charpentier (2012) – in vitro cleavage of specific DNA sequences:

 Designed complementary gRNAs

 Purified Cas nuclease
 Target sequence (20 nucleotides followed by NGG)

Feng Zhang (2013) – edited cultured mammalian cells

 Expression vectors carrying Cas9 gene and gRNA to a specific genomic DNA
target
 Relied on NHEJ or HDR (+ donor template) to repair DSBs
 Created nickases and dCas9
o Dead Cas9 - a modified version of Cas9 where its nuclease activity has
been disabled but it can still bind to specific DNA sequences guided by a
gRNA
 Multiplexing of gRNAs
  CRISPR/Cas fidelity – o -targets

EXPANDING THE CRISPR TOOLBOX (more than just a nuclease)

 Fusing dCas9 to:

o Transcriptional activator – activate the transcription of any target gene
o Transcriptional repressor – repress the transcription of any target gene
o Epigenetic modifier (DNA methyltransferase) – addition of epigenetic tags
o Fluorescent protein (GFP) – visualize the exact position of a target
sequence (molecular paint)
 Base editors
 Prime editors
APPLICATIONS AND POTENTIAL CRISPR/Cas TECHNOLOGY

In the few years since Cas9 was first combined with

exogenous gRNA, CRISPR/Cas has been used for targeting
single loci in a multitude of fungal, bacterial, plant and
animal species, including humans and multiplexed targeting
has been shown in various systems. CRISPR/Cas
technologies have been adapted to diverse applications
including gene activation and silencing, visualising genome
dynamics, genome wide functional screens and diagnostics.

Genome wide association studies (GWAS) have already identified at least 2000
mutations associated with around 300 genetic conditions. CRISPR/Cas should facilitate
multiplex assays to interpret the function of each mutation to further understand and
better treat these conditions.

CRISPR/Cas has applications in many di erent fields such as medicine, biotechnology

and agriculture.

APPLICATIONS IN MEDICINE

 Can be used to disrupt protein coding genes as well as non-coding elements

(regulatory sequences, microRNAs and long non-coding RNAs)
 Genetic variations like SNPs and structural variations can be created in model
organisms to study the physiological roles of these variations
 CRISPR can be modified to make programmable transcription factors that allow
the activation or silencing of specific genes
 Can be injected directly into one-cell embryos to create knock-out or -in animals
(bypasses the use of embryonic stem cells)
 Gene correction in patient-derived stem cells or somatic cells using CRISPR/Cas
can provide novel therapeutic opportunities for patients with diverse genetic and
acquired diseases.
o Genetic correction has been achieved in human cells that were derived
from patients with genetic diseases such as Duchenne muscular
dystrophy, cystic fibrosis, sickle cell disease and Down syndrome
o Removed retrotranscribed simian immunodeficiency virus sequences
from a living primate
o Removed retroviruses from the pig genome to make their organs safer for
transplantation.
 Cas9-mediated targeting of viral RNA in eukaryotic cells – Cas9 can be directed
by an engineered RNA targeting guide to target and inhibit viruses.
  CRISPR was used to repair a genetic mutation responsible for retinitis
pigmentosa (leads to blindness).
 Human genome editing has been done
o CRISPR twins (OCT 2018) who are resistant to HIV infection through the
use of a CCR5 mutation.
o Ethics
 Ex-vivo example: CRISPR sickle cell disease gene therapy
 In-vivo example: Leber congenital amaurosis

APPLICATIONS IN AGRICULTURAL BIOTECH

 Used to increase the crop yield, nutritional enhancement and drought and stress
tolerance as our population grows exponentially.
 Pest, herbicide and disease resistance
 Improved shelf-life
 Reduction of allergens and toxins

CRISPR ANIMALS
  Cattle
o Altered (humanized milk) – CRISPR modification to produce milk that is
similar to human breast milk (allergens and lysozymes)
o Bovine tuberculosis resistance
o Myostatin knock-down (increased muscle growth)
 Pigs
o Removal of 62 di erent retrovirals so that pig organs are more suitable for
xenotransplantation
o In pigs that had GH overexpressed, they gained mass 13.7% faster and
had 18% more e icient feed conversion.
o CRISPR based castration to reduce aggressive behaviour and to avoid the
accumulation of hormones that lead to the boar taint taste and odor.
 Sheep and goats
o Increase in yellow fat by inhibition of beta carotene oxygenase 2
o Myostatin knockdown
 Chickens
o Hypo-allergenic eggs using knockout of egg-allergens ovalbumin and
ovomucoid
 Lab-grown meat: creating real chicken and beef tissue using CRISPR

Lecture 6: Executing a Ged project

ADVANTAGES OF CRISPR/Cas

 Simplicity – targeting by ZFNs and TALENs requires protein engineering which is

complicated. CRISPR/Cas only requires the design and synthesis of a guide RNA
 Super cheap
 Does not require exogenous restriction enzymes
 Low cytotoxicity
 Speed – CRISPR targeting constructs can be injected directly into the mouse
zygote enabling the birth of modified mice just three weeks later.
 Can cleave methylated DNA (ZFNs and TALENs cannot)
 Multiplexed mutations: more than one gene can be targeted at once
 Can di erentially target either DNA or RNA

DISADVANTAGES

 In spite of a 20nt recognition site, non-specific (o -target) cuts do happen.

 Foreign DNA can sometimes be inserted accidently
 Delivery systems (di icult to deliver)
 DSB repair (such as NHEJ and HDR) is random and error prone.
EXECUTING A CRISPR PROJECT: GENOME EDITING

Purpose: to deliver Cas9 and gRNA into a cell

Strategy (bioinformatics)

 Target ID (know the target sequence)

 Multiplexing (targeting multiple sites in the genome simultaneously)
o Allows for the editing of multiple regions within a single gene
o Pathways in cells often involve multiple genes working together, so being
able to edit several of these at once allows for a deeper understanding of
how complex biological processes are regulated.
 If the gene you want to target is an essential gene and it can’t be knocked out it is
possible to edit the REGULATORY ELEMENTS and up or down regulate the gene
instead.
 gRNA design (can be designed with the help of programs such as CRISPR-P and
RGEN)
o PAM: The presence of a correct PAM sequence near the target site is
essential for Cas protein recognition and cleavage.
o Secondary Structure: The gRNA’s folding pattern a ects its ability to bind
to the Cas protein and the target DNA.
o GC Content: The proportion of G and C nucleotides impacts the strength
and stability of gRNA-DNA binding.
 Delivery system (how to get it into the nucleus)
Vector construction (molecular biology)

 AddGene is a plasmid archive where researchers can share and obtain

backbone vectors to use.
 Delivery can be done ‘all-in-one’ (might lead to a too large plasmid which will be
rejected by the nucleus) or using separate vectors (more complicated).
 Promotors (determines how the gene of interest will be expressed)
o determined by the organism the vector is being put into.
 Selection: the process of identifying and isolating cells that have successfully
taken up the vector containing the gene of interest
 Cloning strategy: the method used to insert the gene of interest into the vector.
The main strategies include Gibson assembly and golden gate cloning.

Delivery

 Strategy for Cas

o Expressed from a plasmid under the control of an appropriate promoter
o In vitro transcribed capped mRNA
o Purified recombinant Cas9 protein
o Constitutively expressed in a transgenic organism
 Strategy for gRNAs
o Same or di erent plasmid vector to Cas
o Virus vectors (infect plant)
 Using ribonucleoprotein complexes (DNA free)
o Biolistics
o Protoplasts (chemically transfect a plant cell)
o Inject

Transformation/regeneration of mutated cells (tissue culture)

 Transformation refers to the process of introducing foreign DNA into cells leading
to the incorporation of new genetic material.
 Regeneration: after transformation, the cells must be regenerated into whole,
functioning organisms.

Hardening o

 This refers to the process of genetically engineered (lab-grown) plants gradually

acclimating to natural or greenhouse conditions.

Post-mutational analysis

 PCR – used to amplify the regions of interest for analysis

  Sequencing – sequencing is used to confirm the mutation and check for
unintended mutations. TIDE and ICE are types of sequencing that allow the
analysis of indels.
 Phenotype is analysed to evaluate the e ects of mutations and can be used to
link genotype to phenotype.

EXECUTING A CRISPR PROJECT: CRISPR BASED DIAGNOSTICS

(gone over in detail in lecture 8)

1.) Nucleic acid extraction

- Choose which to extract: DNA or RNA
- QC and quantification (quality control)
- Crude extraction (extracted without purification)
2.) Preamplification
- (RT)-qPCR if working with RNA, PCR if working with DNA
- LAMP (loop mediated isothermal amplification)
- RPA (recombinase polymerase amplification)
3.) CRISPR/Cas assay
- Selection of Cas nuclease
- Selection of reporter (used to detect the activity of Cas protein)
- One pot (combine all steps into one reaction vessel).
4.) Read-out (make it visible)
- Fluorescence
- Lateral flow device (such as the SHERLOCK and DETECTR systems).
- Electrochemical
 Lecture 7: alternative uses of Cas nucleases
Cas nucleases can be used to edit without creating a double stranded break such as in
base and prime editing. They can also be used in CRISPR-based diagnostics and
biosensing (Cas12, 13 and 14 based detection systems such as SHERLOCK, HOLMES
and DETECTR).

BASE EDITING (BE)

 Base editing converts a target base or base pair into another (e.g. A:T to G:C)
without causing DSBs and thus not requiring DSB repair mechanisms.
 Base editors use CRISPR components (gRNAs and Cas9 or other nucleases), but
don’t cut the double helix. Instead, they use deaminase enzymes to chemically
alter single bases.
 You can get base editors for both RNA and DNA

[Grab your reader’s attention

with a great quote from the
document or use this space to
emphasize a key point. To place
this text box anywhere on the
page, just drag it.]
Highly error prone More precise than NHEJ but still
not very e ective unless occurring
in actively dividing cells

DNA BASE EDITORS

 Base editors consist of a fusion of a partially inactive nuclease (nickase) and a

catalytically active base-modification enzyme (deaminase) that acts only of
single-stranded DNAs
 Two types of DNA editors have been developed: cytosine base editors (CBEs)
and adenine base editors (ABEs).
A – In the normal Cas9 nuclease there are two
subunits (RuvC and HNH) that create blunt ended
cuts in the DNA strand. Each subunit cuts through a
single strand, together forming a DSB.

B – An induced mutation in Cas9 means that RuvC is

not actively expressed and only the HNH subunit is
active, cutting a single DNA strand.

C – An induced mutation in Cas9 means that HNH is

not expressed and only the RuvC subunit is active,
cutting a single DNA strand.

CYTOSINE BASE EDITORS

 CBEs are fusions of cytidine deaminases to CRISPR-Cas nickases and can

e iciently induce C to T alterations in DNA
 Uracil glycosylase inhibitor (UGI) help improve the yield of conversion.

ADENINE BASE EDITORS

 ABEs are fusions of adenine deaminases to CRISPR/Cas nickases.

 Causes A to I (inosine) modifications
o Inosine (I) is treated like G by polymerases
RNA BASE EDITING

 RNA base editing enables the direct alteration of RNA molecules rather than
DNA.
 The process is temporary and reversible as RNA naturally degrades and is
replaced over time
 RNA base editing enables the alteration of the function or structure of proteins
without modifying the genome itself.
 RNA base editing typically involves
o gRNA (a ss-RNA that directs the editing enzyme to a specific location of
the target RNA)
o A deaminase enzyme such as ADAR (adenosine deaminase acting on
RNA) that catalyses the chemical change at the target base
o dCas13, fused to the ADAR.
 The two main types of RNA base editing are:
o Adenosine to Inosine (A to I) editing: ADAR enzymes convert adenosine (A)
in RNA to inosine (I) which is interpreted as guanosine (G) by the cellular
machinery.
o Cytosine to Uridine (C to U) editing: ADAR2 enzymes convert cytosine (C)
to uridine (U).

PRIME EDITING (PE)

Prime editing eliminates many of the limits of CRISPR that have caused bottlenecks in
its therapeutic and biotechnological applicability. It is mainly used to fix mutations that
turn normal codons into stop codons (changing protein structure and/or function).

CRISPR causes an injury (a nick) in DNA which needs to be repaired, relying on error-
prone endogenous repair systems. The PE system retains CRISPRs targeting specificity
but also has an edit-containing RNA template as a contiguous extension of the gRNA
(known as the pegRNA) and reverse transcriptase (RT) fused to the C terminus of a
Cas9 nickase. The Cas9 nickase cuts the non-complementary strand of the DNA
exposing a DNA flap with a 3’ OH group which binds to the primer binding site of the
RNA template, serving as a primer for the reverse transcriptase which extends the 3’ flap
by copying the edit sequence of the pegRNA.

[Link]
PE IN ACTION

1. The Cas9-pegRNA complex binds to the

desired target region and creates a nick
3bp upstream of the PAM site. The nick
must be upstream of the first variant site
and occurs on the same strand as the
PAM, liberating a 3’ flap.
2. The 3’ flap forms a sequence specific
interaction with the 14-16 nucleotide
primer binding site located at the 3’ end
of the pegRNA. This RNA/DNA hybrid
serves as the primer site for new DNA
synthesis using the RNA ‘edit site’ as a
template. The RT polymerase copies the
template extending the 3’ flap.
3. The edited 3’ flap displaces the variant
unedited 5’ flap which is removed by
cellular nuclease FEN1.
 Lecture 8: CRISPR-based diagnostics (Dx)
This is a 4-step process comprising of sample extraction, pre-amplification, CRISPR
assay and readout.

SAMPLE EXTRACTION

 Nucleic acid extraction for either DNA or RNA

 Protocols are RNA or DNA specific and mostly incorporated in commercial
extraction kits, optimised for various sample types
 Extracted nucleic acid samples are typically analysed for purity, quality and
quantity before use in assays
 Crude extractions are preferable (extracted without purification)

PRE-AMPLIFICATION STRATEGIES

 Most CRISPR-based diagnostics using Cas without preamplification of the target

reach an LOD in the picomolar range which is good enough for detection when
there is a relatively high concentration of DNA or RNA in the sample but for many
applications, pre-amplification is essential.
 PCR based methods became redundant because of the thermal cycling required
(expensive, time consuming and labour intensive).
 PCR has been replaced by isothermal amplification systems like recombinase
polymerase amplification (RPA) or loop-mediated isothermal amplification
(LAMP).
 RPA (and RT-RPA)
o Uses three enzymes: recombinase, a single strand DNA binding protein
and a strand displacing polymerase
o The recombinase forms complexes with primers and pair these with their
homologous sequences in the target DNA
o DNA synthesis then starts with the strand-displacing polymerase
o RPA steps can all be performed at a constant optimal temperature of
37 degrees Celsius
  LAMP (and RT-LAMP)
o Uses a polymerase with high strand displacement activity and replication
activity
o Uses lots of primers to increase specificity and an additional pair of loop
primers to accelerate the reaction.
o The target sequence is amplified at a constant temperature of 60-65
degrees Celsius which can be achieved in a water bath.
 When CRISPR-based methods are isothermal, they are easy to incorporate into
point-of-care (mobile) devices.

TYPE V AND VI CAS NUCLEASES

Cas9 (type II)

 The first diagnostic systems were based on Cas9

 More complex and so is now redundant

Cas12 (type V), Cas13 (type VI) and Cas14

 These Cas’ possess a unique ability to unleash

indiscriminate (collateral) cleavage of ssDNA and ssRNA
upon target recognition (ds/ssDNA and ssRNA).
 This ability can be exploited for use in diagnostics where recognition of specific
nucleic acid sequences can be easily linked with a readout through the
indiscriminate cleavage activity.
 Cas14 is very similar to Cas12

CAS12 AND CAS13 BASED DIAGNOSTIC SYSTEMS

Cas12-based: DETECTR

 DNA endonuclease targeted CRISPR trans reporter

 Guided to ds/ssDNA targets by a complementary RNA,
triggering collateral cleavage of short ssDNA reporters
carrying a fluorophore and a quencher.
 Target recognition and reporter cleavage lead to the
separation of the quencher from the fluorophore which
generates a fluorescence signal

Cas12-based: HOLMES

 One-hour low-cost multipurpose highly e icient system

 Uses PCR as preamplification together with LbCas12a
 HOLMESv2 uses LAMP combined with a thermostable Cas12b in a one-tube
reaction.
Cas13-based: SHERLOCK

 Specific high-sensitivity enzymatic reporter unlocking)

 DNA or RNA is isothermally amplified with recombinase polymerase
amplification (RPA) or reverse transcription RPA (RT-RPA).
 Uses a forward primer that adds a T7 promoter to the amplicon.
 Allows for RNA transcription of the target, which is then recognized and bound by
Cas13a and a target-complementary (specificity) crRNA.
 Cas13 will then cleave both the on-target ssRNA by cis cleavage and, in a target-
independent manner, the ssRNA reporter molecules by collateral trans cleavage.
 The ssRNA reporter molecules consist of a fluorophore and a quencher joined
together by a short RNA oligomer, which, once cleaved, allows for the separation
of the fluorophore from the quencher, resulting in fluorescence.

SHERLOCK V2

 Quantitative multiplexed detection of nucleic acids (test for 4 di erent

pathogens in one go).
 Uses four Cas12 and Cas13 enzymes each having a unique, specific cleavage
site, combined with uniquely linked quenched fluorescent reporters.

READ OUTS AND QUANTIFICATION

 One tube fluorescence takes advantage of RNA or DNA reporter molecules

carrying a fluorophore and a quencher.
o Collateral cleavage activity on target detection causes the special
separation of the fluorophore and quencher – the resulting fluorescence
can be read on conventional plate readers, cell phones and w=even with
the naked eye under blue light.
 Quantification
o While the target is being amplified, the Cas nuclease is simultaneously
cleaving the reporter molecules due to the collateral cleavage e ect.
o The cleavage of the reporter molecules during amplification generates a
visible signal proportional to the amount of target sequence present.
o This enables real-time readout
o Requires more sophisticated lab equipment
 Lateral flow devices (based on lateral flow immunochromatography)
o Visualisation of the collateral cleavage activity when reporter molecules
carrying biotin and a fluorescein dye (FAM), bind to anti-fluorescein (anti-
FITC) antibodies that are coupled to gold nanoparticles.
 o These complexes subsequently travel to a capture line, where they are
bound by the biotin of the reporter molecule.
o In the absence of the target, uncut reporter molecules are retained there,
and this is visualized as one band on the strip.
o In the presence of the target, Cas-enzyme-mediated collateral cleavage
of the reporter separates the biotin from the FAM molecules bound to the
anti-FITC gold nanoparticles, allowing them to travel farther on the strip
and generating a second visible band at the antibody-capture line, which
bears species-specific secondary antibodies.

 Microfluidic chips (based on electrochemistry)

CRISPR-based biosensing

CRISPR can be used to detect anything that DNA can be attached to. Good for the
detection of heavy metals.

Lecture 9: GEd regulation and ethical issues

I asked what would be examinable from this lecture and he basically said that nothing is other than the
SDN categories. He also recommended having a general idea on the topic of the changing regulations
with GEd and GMOs. What I’ve put in here is information that I think would be useful for an opinion-based
question on GMO and GEd regulation.

SITE-DIRECTED NUCLEASE (SDN) CATEGORIES

Genome-editing is categorised into three types for the sake of regulation:

SDN-1 – introduction of single point mutations or InDels

SDN-2 – short insertions or editing of a few base-pairs by an external DNA-template

sequence

SDN-3 - insertion of longer strands of transgenes or cisgenes.

REGULATION

 GMOs are a highly debated topic as when you put genes from one species into
another you don’t know how these genes will react in the new organism.
o They may cause allergenicity or toxicity and as such testing must be done
to ensure that GMO products are safe.
o There has not been a single case of serious disease or death related to
GMOs in the 27 years that they have been around.
 The problem with GEd products is that they are impossible to test for. You can’t
tell the di erence between a directed mutation caused by CRISPR and a natural
mutation.
 LMO’s – living modified organisms (as opposed to GM products)
 Definition of a GMO according to WHO – organisms in which the genetic material
(DNA) has been altered in a way that does not occur naturally by mating and/or
natural recombination.
o This definition used the PROCESS as a trigger not the end product and
also does not include GEd.
 Most progressive countries define a GMO as an organism of which the genome
has been modified to contain foreign (transgenes) or additional (cisgenes)
genetic material. This definition focuses on the product and not the process.
 South Africa is basically the only country left that considers GEd products GMOs
(Europe and New Zealand having recently changes regulation laws).
THE SECURE RULE

 SECURE – Sustainable, Ecological, Consistent, Uniform, Responsible, E icient

 This rule exempts certain categories of engineered plants from the regulations
because the USDA deems that they could otherwise have been developed
through conventional breeding
 These plants are “unlikely to pose an increased plant pest risk compared to
conventionally bred plants”.
 Examples of exemptions
o Deletions of any size
o Targeted substitutions of a single base pair
o Introductions from sequences derived from the plants natural gene pool
(cisgenes)
o Edits from sequences which are known to correspond in the plants
natural gene pool (intragenics)
o Presence of transgenes previously evaluated and found to be unlikely to
pose a plant pest risk.

GENE DRIVES

This process includes the conversion of a heterozygous individual of a certain trait to

homozygous ensuring that nearly all o spring will inherit the trait. When these traits are
lethal genes, this drive can lead to the elimination of a species within 4 generations.

If reversed this process could possible be used to resurrect species.

 Lecture 10: Case study I
The lecturer said that we don’t need to know any specific details from the case studies (won’t need to
interpret graphs of give bacteria names). We need to understand how these technologies are applied to
solve a problem. She said we may be given a question where there is a problem and we need to apply the
theory we learnt in previous weeks to solve it (like is done in the case studies). I will given short
descriptions of the case studies but I think the best thing to do would be to go over the slides once or
twice to understand how they apply technologies and make sure you are able to do it.

CRISPR/Cas for detection of Cadidatus Liberibacter asiaticus in citrus.

This is bacteria that causes citrus greening, which is a devastating disease a ecting
citrus production. There is no cure and so it must be prevented.

In this case CRISPR/Cas diagnostics are used to detect pathogen (bacteria) levels in
citrus using the CRISPR/Cas12 DETECTR system. Read-out was done using
fluorescence.

Lecture 11: Case study II

Studies indicate that climate change will exacerbate drought events in many traditional
grape growing regions which will have significant negative impacts on the productivity of
vineyards. It is important that we identify the key traits for the development of new
drought-tolerant varieties.

Drought and stomata

Stomata are pores located in the leaf epidermis. The opening of these pores controls
leaf gas exchange. Stomata are key players in a plant’s response to drought. The genetic
manipulation of stomatal activity is a promising approach to reduce the water
requirement of crops and to enhance productivity

Abiotic stressors are stressors that are environmental rather than caused by pathogens
(such as drought, wind and nutrients).

MYB60

In Arabidopsis, atMYB60 has been linked to stomatal regulation. Arabidopsis myb60

knockout mutants display constitutive reduction of light-induced stomatal opening and
enhanced tolerance to drought. VviMYB6p is the gene encoding the grapevine ortholog.

To avoid undesirable side e ects on plant growth and productivity, a promoter could be
a better target for genome editing. Thus, it would be better to target the DOF clusters
(transcription factors) that regulate MYB60 expression.
Editing analysis

There are two methods of editing analysis: Sanger and Illumina. Sanger sequencing is
cheap and easy (good for first screenings) but it is super precise when it comes to
determining which edits and mutations have come about. Illumina is more precise and
can be used along with CRISPResso to analyse the outcome of genome editing (tells
you what mutations have occurred where). In most cases sanger is done first to
determine if the edit was successful and then illumina to see what edit occurred.

Case study II

In this case study CRISPR/Cas was used to target the promotors of a stomatal pore
opening gene. Sanger and Illumina were used to determine the outcome of the edit
which was hypothesized to be reduced stomatal pore opening. However, this was not
the case. Next drought stress tests will need to be done to see if the new phenotype will
only come about in stress conditions.

The problem with CRISPR/Cas is that it relies on natural DBS repair technology and so
mutations cannot be controlled. In this case study the common mutation did not
change the gene. Base editing may be a better system as precise gene edits can be
made.

Lecture 12: Case study III

 Viticulture is a sector with one of the highest uses of fungicides
 The use of pesticides is becoming increasingly unsustainable in terms of cost,
human health and environmental concerns.
 To substantially reduce chemical treatments, one approach is to breed resistant
plants that possess a wide range of resistances. This can be achieved by
introducing resistant loci.
o Resistance is quickly overcome by pathogens
 New strategies focus on modification of susceptibility (S) genes
o S genes: host plant genes that promotes compatible interactions with
pathogens (necessary for the pathogen to infect the plant).
o Using CRISPR/Cas knock-out editing.

DMR6 and DLO genes are an example of S genes. They regulate salicylic acid (convert it
into an inactive form) which has a negative e ect on immunity. These genes are
upregulated in pathogen infection. In sequencing of these genes it was found that DLO
genes group together and DMR6 genes group together making them easy to target.
CRISPR/Cas9 editing of these genes could help reduce pathogenicity of certain
diseases.

gRNA design - BLASTN is used to find guide RNA sequences (regions within these genes
next to PAM sequences). We can use multiplex to target multiple genes at once.

Vectors are made with CRISPR/Cas genes and transformed into the grape vine using
agrobacterium-mediated transformation.

The read-out techniques discussed in the previous case study can then be used to
determine if and what editing has taken place.