Transcript RNA-guided endonuclease provides a therapeutic strategy to cure

정동진,유형구,전용호,안은성,김지민
Contents
introduction
A Natural Model of Latent EBV Infection
EBV Genome editing
How can confirm the effect of sgRNA?
Discussion
Introduction
 herpesviridae virus infection problem
- herpes virus subtypes infect a wide range of cells
- herpes virus infection caused a serious disease like cancer, HIV
- herpes virus is not possible to find therapeutic approaches that
completely erdicate
- herpes virus treatment is Acyclovir Famciclovir
- but these treatments have a problem like a side effect, high cost
HOW?
• viral genome-specific nucleases (CRISPR)/Cas9 is a
naturally occurring bacterial immune system
• DNA sequence recognition requires
- single 20-nt guide RNA
- PAM(protospacer adjacent motif)
A Natural Model of Latent EBV Infection
 EBV was initially isolated from cultured
Burkitt’s lymphoma samples.
 Raji cell
 The close relationship between the Raji EBV
genome and the EBV reference.
Raji cell
- First continuous human cell line from hematopoietic
origin
- The cell lines produce an unusual strain of Epstein
Barr virus which will both transform cord blood
lymphocytes and induce early antigens in Raji cells.
- Use for detection of immune complex because it
possesses and expresses and lots of receptors for
certain complement components, as well as Fc
receptors for immunoglobulin G.
Desing of EBV-Targeting CRIPR/Cas9
 Plasmids consisting of a U6 promoter-driven chimeric
guide RNA (sgRNA) and a ubiquitous promoter-driven
Cas9 were obtained from Addgene
The type II prokaryotic CRISPR/Cas adaptive immune
system has been shown to facilitate RNA-guided sitespecific DNA cleavage.
- We engineered two different type II CRISPR/Cas systems
and demonstrate that Cas9 nucleases can be directed by
short RNAs to induce precise cleavage at endogenous
genomic loci in human and mouse cells.
- Cas9 can also be converted into a nicking enzyme to
facilitate homology-directed repair with minimal mutagenic
activity.
-
* HDR is a mechanism in cells to repair double strand DNA lesions.
Desing of EBV-Targeting CRIPR/Cas9
 Lymphocytes are known for being resistant to lipofection.
*Lipofection(or liposome transfection) is a technique used to inject genetic
material into a cell by means of liposomes.
 Used nucleofection for DNA delivery into Raji cells
 Chose the Lonza pmax promoter to drive Cas9
 Obvious EGFP signal from a small proportion of cells
 The EGFP cell population decreased dramatically after that,
however, and we measured <10% transfection efficiency 48 h after
nucleofection
* Nucleofection refers to electroporation, a transfection method which
enables transfer of nucleic acids such as DNA,RNA,Small interfering RNA
into cells.
 Attributed this transfection efficiency decrease to the
plasmid level with cell division.
 Redesigned the CRISPR plasmid to include the EBV
origin of replication sequence, oriP.
 Active plasmid replication inside the cells, the
transfection efficiency rose to >60%
 We targeted six regions with seven guide RNA
designs for different genome editing purposes.
 EBNA1 is crucial for many EBV functions, including
gene regulation and latent genome replication.
*EBNA1 is a multifunctional, dimeric viral protein
associated with EBV.
*It is the only EBV protein found in all EBV-related
malignancies.
 We targeted guide RNA sgEBV4 and sgEBV5 to
both ends of the EBNA1 coding region to excise this
whole region of the genome.
 EBNA3C and latent membrane protein-1 are
essential for host cell transformation, and we
designed guide RNAs sgEBV3 and sgEBV7 to target
the 5′ exons of these two proteins, respectively.
EBV Genome editing
•The double-strand DNA breaks generated by CRISPR are
repaired with small deletions.
•These deletions will disrupt the protein coding and hence create
knockout effects
•Beyond the independent small deletions induced by each guide
RNA, which disable individual genes, large deletions between
targeting sites can systematically destroy the EBV genome.
Guide RNA sgEBV2 targets a region with 12 125-bp repeat units (Fig. 2A). PCR amplification of
the whole repeat region gave an ∼1.8-kb band (Fig. 2B).
After 5 or 7 d of sgEBV2 transfection, we obtained ∼0.4-kb bands from the same PCR
amplification (Fig. 2B).
We further demonstrated that it is possible to delete regions between unique targets(Fig. 2C)
Six days after sgEBV4-5transfection, PCR amplification of the whole flanking region
(with primers EBV4F and -5R) returned a shorter amplicon, together with a much fainter band
of the expected 2 kb (Fig. 2D).
Sanger sequencing of amplicon clones confirmed the direct connection of the two
expected cutting sites (Fig. 2F).
A similar experiment with sgEBV3-5 also returned an even larger deletion,
from EBNA3C to EBNA1 (Fig. 2 D and E).
How can confirm the effect of sgRNA?
 Cell proliferation arrest
 Genome destruction
 Clearance of subpopulation
Cell proliferation arrest
 Concept
EBV infected cells(Raji)
[lysogenicity]
Suppressing
Increase the EBV
the EBV multiplication
proliferation
Targeted gRNA
(sgEBV)
Cell proliferation arrest
 Procedure
CRISPR
transfection
Culture
(2d after)
cell count
(e.d)
-Two days after CRISPR transfection, further culture and
count the live cell everyday.
-Raji cell is cultivated in 37 degree incubator. (5%CO2)
[media : RPMI-1640(10%FBS)]
Cell proliferation arrest
 Results data
* Results
Untreated - control
Cas9
- lost EGFP express in few days
Cas9-oriP - maintain EGFP expression
sgEBV
Cell proliferation curves after different CRISPR
treatments. Five independent sgEBV1–7 treatments
are shown here.
- no proliferation
constant or decreased(count)
Cell proliferation arrest
 Cytotoxicity test
Raji cell
DG-75
sgEBV targets to specific cells infected by EBV. Only.
IMR-90
Genome destruction
 Concept
Restore
Interruption of apoptosis
the apoptosis process
Host cell(Raji)
EBV infection
This test measures the efficiency of sgEBV(gRNA) through the degree of apoptosis
Genome destruction
 Procedures
1. Examination of apoptotic cell morphology
(microscopic & flowcytometry)
2. Staining of special dye
Genome destruction
 Examination of apoptosis cell morphology
(using the microscopy)
Microscopy revealed apoptotic cell
morphology after sgEBV1–7 treatment.
Nuclear morphology before and after
sgEBV1–7 treatment.
Genome destruction
 Examination of apoptosis cell morphology
(using the flowcytometry)
-Shranking the cell size
-Increasing granulation
» a sign of apoptosis
before
5days
8days
Flow-cytometry scattering signals before,
5 d after, and 8 d after sgEBV1–7 treatments.
X axis : forward scatter
Y axis : side scatter
Genome destruction
 Staining of special dye
( using DAPI and Annexin V alexa647)
before
5days
X axis : Annexin V alexa647
Y axis : DAPI
8days
Clearance of subpopulation
 Procedures
1. Observation of gene copies
2. Real-time PCR
Clearance of subpopulation
 Observation of gene copies
Clearance of subpopulation
 Real-time PCR
Before treatment
After treatment
Discussion
 Transfection of a guide RNA mixture.
- cell proliferation, apoptosis pathway.
- 25%, 50% of cells.
 The availability of viral genomes as therapeutic targets.
- Herpesviridae
- HSV model
- EBV model
Discussion
 CRISPR/Cas9 system
- Cas 9 enzyme + guide RNA
- Off target cleavage : single molecular microscapy
immunoprecipitation assay
 Destroying the entire EBV genome using guide RNAs
targeting several repeat regions.
- drive host-cell proliferation
- Best result
Further story
 Sophisticated approaches to delivery for treatment.
 Delivery approach
- ex) HSV-1, HSV-2, EBV
 Engineer delivery virus.
 Design targeted delivery vehicles
 The delivery material directly.