WEEK 7:Gene Manipulation

Dr. ZuFu Lu
[email protected]
Tissue Engineering &
Biomaterial Research Unit, The University of Sydney
 What is genetics?

Genetics is a term that refers to the study of genes and

their roles in inheritance - in other words, the way that
certain traits or conditions are passed down from one
generation to another. Examples of genetic or inherited
disorders include breast cancer, cystic fibrosis,
Huntington's disease, and phenylketonuria.
Human Genome Project

The Human Genome

Project (HGP) :
is an international scientific
research project with the
goal of determining the
sequence of chemical base
pairs which make up human
DNA, and of identifying and
mapping all of the genes of
the human genome from
both a physical and a
functional standpoint.
 Mission of HGP

› The Mission of the HGP: The quest to understand the

human genome and the role it plays in both health and
disease.

“The true payoff from the HGP will be

the ability to better diagnose, treat,
and prevent disease.”
--- Francis Collins, Director of the HGP and the
National Human Genome Research Institute (NHGRI)
 The Completion of the Human
Genome Sequence

› Publication of 90 percent of the

sequence in the February 2001
issue of the journal Nature.

› Completion of 99.99% of the

genome as finished sequence on
July 2003.
› About 27,000 gene identified.
 Challenge of HGP?

Identification of gene function

› proteins that make up physical components.
› Function(walking, digestion, fighting disease).

OR

Eye Color Hair Color

 Gene manipulation

Critical tool for gene function identification

› Down-regulation
› Up-regulation.
Strategies for gene
manipulation
 Gene manipulation at
genomic level
Promoter: a region of DNA that initiates transcription of a particular gene.
Promoters are located near the transcription start sites of genes, on the
same strand and upstream on the DNA, can be activator or repressor.

Transcription factor: (sequence-specific DNA-binding factor) is

a protein that binds to specific DNA sequences, thereby controlling the
rate of transcription of genetic information from DNA to messenger
RNA

Transcription
factor
 Construction of controllable
genes
Transcription Transcripted sequences
Known factor 1 to mRNA 1
Gene

Transcription Transcripted
factor 2 sequences to mRNA 2
Unknown
Gene 2
 Construction of controllable
genes
New gene
construct
Transcription
factor 1 Transcripted
sequences to mRNA 2
 Induced pluripotent stem cells
(iPS)

Somatic (adult) cells reprogrammed to enter an embryonic

stem cell–like state. Similar properties to embryonic stem
cells as can differentiate into many different tissue types –
pluripotent
RNA interference technology
(RNAi)
Also Called PTGS, for “Post mRNA
Transcriptional Gene Silencing”
 RNAi terms

› dsRNA: double stranded RNA, longer than 30 nt

› miRNA: microRNA, 21-25 nt.

- Encoded by endogenous genes

› siRNA: small-interfering RNA, 21-25 nt.

- Mostly exogenous origin
 What is RNAi?

› “The Process by which dsRNA silences gene expression...”

› Degradation of mRNA or translation inhibition
 Discovery of RNAi

Sense RNA: a single strand of DNA sense (or positive

(+)) if an RNA version of the same sequence is translated
or translatable into protein

Antisense RNA: Antisense RNA is an RNA transcript that

is complementary to endogenous mRNA

Sense RNA
 Discovery of RNAi

Principle of antisense RNA strategy for

blocking gene expression

Sense RNA

Antisense RNA
 Unexpected results by
using antisense RNA

› Guo and Kemphues studied par-1 gene during embryogenesis

 Craig Mello

› In 1996, C. Mello and his student S. Driver also reported

that sense RNAs mimic antisense phenotype.
 Andrew Fire
› In 1991, A. Fire successfully targeted genes by antisense
constructs from transgenes.
› Sense constructs also exhibited silencing activity.
 Fire and Mello
› Their paths crossed:
- Mutual interest on a gene called pie-1.
- Discuss what the sense and antisense preparations have
in common
 Hypothesis

ssRNA in vitro synthesized by bacteriophage

RNA pol might be contaminated by dsRNA
Bacteriophage RNA polymerase produce random
ectopic transcripts
 Discovery of RNAi

Blocking effects
are resulted from
dsRNA
 Amazing Medical Discoveries
Found By Accident

The pacemaker

Pap smear
 The mechanisms of RNAi
technology (two phases)

› Initiation
- Generation of mature siRNA

› Execution
- Silencing of target gene
- Degradation or inhibition of translation
The mechanisms of RNAi
technology
1. Introduction of ds RNA in
the cell or by artificial
means using vectors or
short hairpin RNA (shRNA)

2. Recognition and
processing of long dsRNA
by Dicer, an RNase III
enzyme

3. Duplexes of siRNA of 21-

24 nucleotides length
formed by Dicer
The mechanisms of RNAi
technology

4. Incorporation of both
synthetic siRNA into RNA-
induced silencing
complex(RISC)

5. Unwinding of duplex
siRNA by a helicase in RISC
and removal of messenger
strand (RISC activation)
The mechanisms of RNAi
technology

6. Recruitment of RISC
along with antisense strand
to target mRNA

7. Cleavage of target mRNA

by an unidentified RNase
(Slicer) within RISC.
Degrades mRNA at sites not
bound by siRNA
 Applications

› Genome-wide RNAi screens

- Gene function
- Candidate genes and drug discovery

› Therapy
- Candidate genes, drug discovery, and
therapy.
siRNA in Bone Regeneration

Large amounts of bone

loss due to trauma, tumor
excision, or infections do
not heal on their own and
require therapeutic
intervention.
 siRNA in Bone Regeneration

Stem cells Bone cells

Bone Implanted Bone
defect scaffold healing

Synthetic bone
substitute
 siRNA in Bone Regeneration

Noggin

Inhibition of Noggin expression in stem cells might increase

BMP signaling pathway and instructing stem cell to become bone cells
 siRNA in Bone Regeneration
The prolonged delivery of siNoggin or
sioggin/miRNA-20a enhanced the osteogenic
differentiation of encapsulated hMSCs.

Biomaterials, 2014, 35(24), 6278-6286

 RNA-targeted therapeutics in cancer clinical trials

Drug Tumor type Target Delivery platform Phase of study Best response Most common or Citations
dose-limiting
toxicity (DLT)
siRNA/dsiRNA
DCR-MYC Advanced Myc Lipid nanoparticles I/II 1 CR, multiple PR 1 DLT = G3 LEE; Tolcher et al.[32]
cancers (n.s.) G1-2 fatigue, ICE
ATN-RNA Brain Tenascin-C Naked molecule I Better overall No significant Rolle et al. [31]
survival, quality of neurotoxicity
life
TKM 080301 Neuroendocrine/ PLK1 Lipid nanoparticles I/II 3% PR, 11% SD Transient G1/2 Ramesh et al.[25]
adrenal nausea/vomiting,
fatigue, ICE
siGD12 LODER Pancreas KRAS LODER polymer I/II 17% PR, 83% SD Transient G1/2 Golan et al.[28]
abdominal pain,
diarrhea, nausea
Atu027 PKN3 Lipid nanoparticles I/II Progression-free 17% G4 and82– Schultheis et
survival not 92% G3 events al. [30]
statistically (n.s.)
different
Atu027 Solid tumors PKN3 Lipid nanoparticles I 41% SD <10% G3 (n.s.), 2 Schultheis et
G4 GGT and al. [29]
lipase elevation
ALN-VSP02 VEGF, KSP Lipid nanoparticles I 8% PR, 47% SD 1 death, 2 DLT = Cervantes et
1 G3 al.[26]
thrombocytopenia
, 1 G3
hypokalemia,
10% ICE
CALAA-1 RRM2 Cyclodextrin I No objective Closed owing to Zuckerman et
nanoparticles responses DLTs: 50% al. [27]
fatigue, 42% fever
 Major limitations of RNAi
technology

Delivery: The biggest problem with the use of RNAi is its

successful delivery to the target. RNAi must be stable in
a cell for prolonged activity without getting degraded.

Off-target inhibition: Non-specific interactions can occur

because, though siRNA can be designed to target a
specific sequence, a difference in one or two base-pairs
is sufficient to cause off-target binding.
 What you need to know?

Understanding of
What is RNAi technology and its
underlying mechanisms
 CRISPR/CAS9 SYSTEM

Clustered regularly interspaced

short palindromic repeats
(CRISPRs) and CRISPR-
associated (Cas) proteins
Publications
Funding
 What is CRISPR-Cas9

 The mechanism of adaptive immunity in

bacterial and archaea to defend against
foreign genetic material (e.g. phage)

 Type II pathway: CRISPR-Cas9, creating a

break in double-strand in the targeted DNA.
Three steps of CRISPR-CAS
action
The first stage, adaptation, leads to insertion
of new spacers in the CRISPR locus.

In the second stage, expression, the system

gets ready for action by expressing the cas
genes and transcribing the CRISPR into a long
precursor CRISPR RNA (pre-crRNA). The pre-
crRNA is subsequently processed into mature
crRNA by Cas proteins and accessory factors.

In the third and last stage, interference,

target nucleic acid is recognized and destroyed
by the combined action of crRNA and Cas
proteins.
 Two Major repair pathways
DSBs

Two types of repair pathways address double-

strand breaks (DSBs)

1. Non-homologous end-joining (NHEJ), which has no

source of information.
2. Homologous recombination (HR): which is to use the
sister chromosome as a source of information,
 Two Major repair pathways

NHEJ HR

NHEJ HR

Uses no template
Requires a homologous template
Creates insertion or the deletion of
(donor)
bases (Indels) and more error-prone
Inefficient, but more accurate
With higher frequency
Application of CRISPR/Cas9
System by NHEJ or HR
 Potential application of
CRISPR-Cas9
Targeting on genomic level
Hypertrophic Cardiomyopathy
(HCM)

 HCM is a hereditary disease. It is

caused by abnormalities in genes
which make the protein which
causes the contraction of the heart .

 The mutation of MYBPC3 gene

causes HCM

 There is no cure for HCM, treatment

given is to prevent complications
and improve symptoms.
 Gene correction in M-phase-
injected human embryos.

CRISPR–Cas9 was co-injected with sperm into oocytes. This

allows genome editing to occur when a sperm contains a
single mutant copy.
Great need for Organ Donation

In Australia, 1,713 people

received organ donations in
2016.
About 1,400 people are
waiting for organ donations
at any one time, with an
estimated 100 Australians
dying on waiting lists each
year.
 The possibility of using pig
organs in xenotransplantation
The organ size is similar to humans, they reproduce
quickly and they can be genetically manipulated to reduce
the risk of rejection.

Limitation:

There are endogenous

retroviruses in the pigs, and
human embryonic kidney cells
became infected when
researchers grew pig cells
next to them.
 Genetically modified pigs as human
organ transplantation source
Using CRISPR-Cas9, the PERVs were inactivated in a
porcine primary cell line and generated PERV-inactivated
pigs via somatic cell nuclear transfer

Science 10 Aug 2017:eaan4187

DOI: 10.1126/science.aan4187
RNAi vs.CRISPR-Cas9
 What you need to know?

Understanding of
what is CRISPR-Cas9 technology and
the difference between RNAi and
CRISPR-Cas9