Group 1(Reena Shama JaeHee Yasmin and Nadha)

1. Aishath Reena
Jung JaeHee
Mariyam Shama
Nur Yasmin
Aminath Nadha
0336105
0332948
0336069
0336968
0336126

2. ● Tripronuclear (3PN) zygotes have one oocyte nucleus and two sperm
nuclei.
● Discarded in clinics, during in vitro fertilization (IVF) clinical trials
● In this study, Tripronuclear (3PN) zygotes are used to investigate
CRISPR/Cas9-mediated gene editing in human cells.
● Used because of ethical concerns regarding gene editing in normal
embryos
● Ideal model system to examine the targeting efficiency and off-target
effects of CRISPR/Cas9 during early human embryonic development
Dispermy

3. Components
- gRNA: guide RNA, used to direct the
Cas9 protein to bind and cleave a
particular DNA sequence for genome
editing
- Cas: CRISPR-associated System,
endonuclease protein; able to cleave
nucleic acid.
- *PAM: protospacer adjacent motif, a short
specific sequence, essential for cleavage
by Cas endonuclease
*NHEJ: Non-Homologous End Joining

4. Objective: To cleave and repair HBB gene coding for protein Hemoglobin
using CRISPR/Cas9 system - can be used to correct mutations which leads
to disorders such as 𝛃-thalassemia.
* 𝛃-thalassemia is a genetic blood disorder (inability to produce RBC)
Application: CRISPR can efficiently cleave the
endogenous gene in human tripronuclear zygotes
(3PN) resulting in double strand break (DSB) which can
be repaired by non-homologous end joining (NHEJ)
and homologous recombination directed repair (HDR).

5. CRISPR
cas9
plasmid
Identification
& collection of
tripronuclear
zygotic
embryos
Embryo
vitrification
and
recovery
Analysis of
CRISPR/Cas9
induced
cleavages
Injection of GFP mRNA
and Cas9/gRNA/ssDNA in
different concentration
combinations into 3PN
zygotes intra-
cytoplasmically
Whole genome
amplification
using Embryos
Whole-exome
sequencing,
data
processing,
and off-target
analysis

6. A)DSBs may be repaired
through the double-
strand break repair (DSBR)
pathway or the non-
crossover synthesis-
dependent strand
annealing (SDSA) pathway
B)The HBD locus from the
7 recombined 3PN
embryos were similarly
examined as above.*
Indicates that the HBD
locus failed to be
amplified in two of the
embryos.
C)Repair of DSBs
generated by
CRISPR/Cas9 occurs
mainly through NHEJ.
If HDR is utilized, the
non-crossover
pathway is preferred.

7. - Of the 54 PCR-amplified embryos, 28 were cleaved by Cas9, indicating
an efficiency of ∼52%
- 4 of the 28 Cas9-cleaved embryos (14.3%) were clearly edited using the
ssDNA oligo as a repair template
- 7 out of the 28 cleaved embryos (25%) shows recombination of the HBB
gene with HBD , even in the presence of co-injected exogenous ssDNA
donor template

8. Advantage Limitation
- Increased Experimental
Convenience
- Less ethical concern as
3PN are discarded in
clinics anyway
- Used as an alternative to
normal human zygote
- Off-target changes leading
to unintended mutations
- Difficult to apply RNA
polymerase Ⅱ for gRNA
production
- Efficiency of delivery
method depends on the
type of target cells/tissues

9. Current developments
● Clinical applications are premature
● Introduction of a naturally occurring allele into early human 3PN
zygotes (Fan, 2016).
Future perspectives
● Need to improve the specificity and accuracy before proceeding to
clinical applications
● Need to understand the mechanisms more comprehensively
● Genetic mutations can be corrected in early embryos at a premature
stage (developing treatments for genetic disorders)

10. Reference
Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., Lv, J.,
Xie, X., Chen, Y., Li, Y., Sun, Y., Bai, Y., Songyang, Z., Ma, W.,
Zhou, C. and Huang, J. (2015). CRISPR/Cas9-mediated gene
editing in human tripronuclear zygotes. Protein & Cell, 6(5),
pp.363-372.
Kang, X., He, W., Huang, Y., Yu,. Chen, Y., Gao, X., Sun, X., Fan,
Y. (2016). Introducing precise genetic modifications into
human 3PN embryos by CRISPR/Cas9-mediated genome
editing. J Assist Reprod Genet, 33(5), pp. 581-588