IARS syndrome in Japanese black cattle causing high motality rate corrected using CRISPR/Cas9 method

1. CRISPR/Cas9-assisted Genome Editing
(Correction of a Disease Mutation using CRISPR/Cas9 in
Japanese Black Cattle)
- Ngan Boon Keat (0333894)
- Beh Yoke Leng (0338291)
- Ng Xian Gui (0337937)
- Chu Sin Ton (0338668)

2. The Disease:
Isoleucyl-tRNA synthetase(IARS) syndrome
Caused by:
Side effects of selective breeding (recessive mutations) to produce high quality meat.
What is it:
A substitution in a IARS gene that causes a reduction in aminoacylation activity in the
IARS protein that impairs protein synthesis.
Effects:
Homozygous mutant calves experience growth retardation in the uterine and more
likely to die before birth.

3. What is CRISPR/ Cas9:
- Stands for "clusters of regularly
interspaced short palindromic repeats"
- Simplest and precise genetic
manipulation tool ----> allowed DNA
sequences alteration and gene function
modified
- Enabled a site-specific double-strand
break to be introduced at a desired locus
in genomic DNA
- Edit short sequence(usually 20-50
codons)

4. How does CRISPR-Cas9 work
4. Cas9 enzyme cuts both strands of
DNA
3. Cas9 enzyme binds to guide RNA
5. The cut is repaired
Introduce mutation
1.Find out the target
sequence
2. The guide RNA is designed to find a
specific sequence in the DNA
Guide RNA binds to target sequence
From (yourgenome.org,2016)

5. Repair of mutant IARS gene
- CRISPR/Cas9-expressing vector
and the donor DNA fragment for
IARS gene repair were introduced
into the BFF cells to form
recombinant cell lines using
electroporation method.
Isolation of BFF cells
- Bovine fetal fibroblast (BFF)
cells obtained from fetuses of
sacrificed cow.
CRISPER/CAS 9 Design
- Design of CRISPR/Cas9 system
system that recognizes the
mutated IARS sequence
- Donor DNA design that is
homologous to the mutation site
of IARS gene
Experiment Outline

6. Success analysis
- Pregnant cows sacrificed on
day 34/46 to retrieve
developing fetuses.
- Fibroblast cells isolated from
fetuses.
- PCR, Sequencing, Southern
blot analysis of genomic DNA
of BFF cells to check for
mutated single nucleotide of
IARS gene.
Isolation of Recombinant BFF cells
- Cells sorted using Aequorea
coerulescens Green
Fluorescent Protein (AcGFP)
expression.
- AcGFP positive colonies were
developed into cell lines.
- Southern blot analysis to check
precise recombination
Generation of Cloned Fetuses
from Recombinant BFF Cells
- Cell lines used as donor cells to
develop embryos using Somatic
cell nuclear transfer (SCNT)
- IARS-repaired BFF cells
transferred into enucleased
oocyte.
- Embryos implanted into recipient
cows.

7. CRISPER/CAS 9 design in this Case Study
1. sgRNA was designed to introduce a double-strand DNA break near the
mutation site in the IARS gene.
2. The protospacer adjacent motif(PAM) sequence was designed and located at
target region.
3. Oligo DNA for the sgRNA was cloned into bacteria vector(pX330)
4. Cleavage activity was performed and estimated by traffic reporter system
5. Electroporation is used to make the cell become competent cell
6. Analyse the expression of EGFP by cell sorter
7. pX330 or pX330_IARS plasmid were introduced into BFF cells with AcGFP
expression vector through Electroporation.
8. Genomic DNA was extracted from AcGFP-positive cells
9. T7E1 assay was performed to produce a series of band

8. Results (Success)
● CRISPR-Cas9 is used to create a double-strand break near the mutation site.
● The result of fetal genomic DNA analysis showed correct repair of the IARS
mutation without any additional DNA footprint.

9. Current development
Whole genome sequencing
● Enabled researchers to identify variants in individuals
● Identify variants from exons and their flanking regions, to identify the causative mutation for
Japanese black cattle disease.
● To perform whole exome sequencing, bovine whole-exon capture custom array (Nimblegen) that
targeted 174,377 exons have been used. It enabled us to identify the causative mutation
responsible for the hereditary perinatal weak calf syndrome.
● Mapping and exome sequencing - identify the mutation responsible for this disease
● Advantages: efficient and cost-effective

10. PROS Cons
To restore genetic diversity Difficult to select donor cells that have
undergone the correct repair (passage
number of the donor cells needs to be
minimal to avoid abnormalities in the
cloned embryo)
To enable the genetic resources of livestock
to be utilized effectively
Cas9 enzymes may cut off wrong gene
Improvement for genetic improvement in
livestock that have a long gestation cycle

11. References
Hirano, T. et al. Mapping and exome sequencing identifies a mutation in the IARS gene as the cause of hereditary perinatal weak calf
syndrome. PLoS ONE 8, e64036, https://doi.org/10.1371/journal.pone.0064036.s003 (2013).
Hirano, T. et al. IARS mutation causes prenatal death in Japanese Black cattle. Animal science journal = Nihon chikusan Gakkaihō
87,1178–1181, https://doi.org/10.1111/asj.12639 (2016).
Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proceedings of the
National Academy of Sciences of the United States of America 93, 1156–1160 (1996).
Christian, M. et al. Targeting DNA Double-Strand Breaks with TAL Effector Nucleases. Genetics 186, 757–761,
https://doi.org/10.1534/genetics.110.120717 (2010).
Yourgenome.org. 2020. What Is CRISPR-Cas9?. [online] Available at: <https://www.yourgenome.org/facts/what-is-crispr-cas9>
[Accessed 3 June 2020].
Science | AAAS. 2020. Beyond CRISPR: What’S Current And Upcoming In Genome Editing. [online] Available at:
<https://www.sciencemag.org/features/2019/09/beyond-crispr-what-s-current-and-upcoming-genome-editing> [Accessed 4 June 2020].