Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9

1. Efficient Production of Biallelic
RAG1 Knockout Mouse Embryonic Stem
Cell Using CRISPR/Cas9
Animal Biotechnology (BIO 61504)
Sylvia Oh Wen Shuen
Siti Rhania Putri Ramadhani
Sharvind Kumar A/L Sunmuganathan
Natasya Valentina Thomas
Natasha Leong Kah May

2. Table of contents
WHAT IS CRISPR/CAS9?
RECOMBINATION-ACTIVATING GENE (RAG)
PROBLEMS & OBJECTIVES
METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

3. WHAT IS CRISPR/CAS9?
Clustered Regularly Interspaced
Short Palindromic Repeats (CRISPR)
and CRISPR-associated protein 9
(Cas9) is a genome editing tool
adapted from a naturally occuring
system in bacteria (NIH, n.d.).
Figure 1. CRISPR-Cas9 with gRNA complex (Walter,
2017)

4. CRISPR-CAS9 mechanism
1. gRNA has a 5′ end complementary to the
target DNA sequence and binds to the Cas9
protein.
2. Conformational change activates protein.
3. When the Cas9 protein finds a potential target
sequence with the appropriate PAM, the
protein will melt the bases immediately
upstream of the PAM and pair them with the
complementary region on the guide RNA
(Sternberg et al. 2014).
4. If the complementary region and the target
region pair properly, the RuvC and HNH
nuclease domains will cut the target DNA,
introducing DSBs (Anders et al. 2014).
5. The DSBs will be corrected view NHEJ
recombination or HDR.
Figure 2. DSBs corrective mechanism (Chegg,
nd).

5. PROS & CONS of CRISPR-CAS9
Table 1. Pros and cons of CRISPR-CAS9
(Bayat et al, 2016).

6. Recombination-activating gene (RAG)
Roles in rearrangement
and recombination of
immunoglobulin and T-
cell receptor genes
during process of V (D) J
recombination
Absence of T-cells
and B-cells
Exhibit impaired
lymphocyte
development
Used as
Immunodeficient
mouse models
(Mehravar et al., 2019)

7. OBJECTIVES
To create desired and specific RAG-
1 -/- mutant mouse in a shorter
period of time
1. Nature of mutations from microinjection
of zygotes cannot be determined prior to
the mouse development.
2. Somatic mosaicism in embryos that
results in allele complexity.
3. Reaching specific mutant or knock-out
gene requires crossing animals which
takes a longer period of time.
1
2
To investigate the nature of
mutations in mutant mESC in order
to achieve complete knock-out of
RAG1 gene.
PROBLEMS
(Mehravar et al., 2019)
3 To compare the sgRNAs sequences
of RAG1 and RAG2

8. Design of specific single nucleotide RNAs (sgRNAs)
METHODS
1
2
- pX330 vector and pLenti-Cas-Guide Vector (sgRNA scaffold and CAs9)
digested with BbsI and BamHI respectively
- Treated with Alkaline Phosphatase (thermo scientific, EL0051)
- Heat and annealing using thermocycler (short double strand DNA
fragment ligated into linearized vector)
Validation of the CRISPR/Cas9 Genome Editing for
Introduction Targeted DSBs
- NIH3T3 cell line was co-transfected with Lipofectamine 2000 and
using GFP plasmid to monitor transfection efficiency.
- Puromycin selection carried out over 3-5 days and genomic DNA
was isolated.
- Amplified the fragments (RAG1 and RAG2) using PCR and
visualize with 1.5-2 % agarose gel
Figure 3. gRNA sequences and RAG gene
sequences.
(Mehravar et al., 2019)

9. RAG1 knockout ES cell production by CRISPR/Cas9 system (clone isolation)
Real Time Reverse Transcription PCR & statistical analysis (SPSS)5
4
- Dilutions were performed across the gelatin-coated 96-well plates
- visualize under microscopy (after 4-5 days)
- clone were transferred into new gelation-coated 96-well plates
- DNA was extracted from ES cell clones and the required regions of RAG1 gene were
extracted and amplified by PCR.
METHODS
- SYBR Green detection
- TRIzol reagent (control)
ES cell culture and transfection3
- Male ES Cells grown under feeder-free culture condition in mESC proliferative
medium supplemented with R2i
- Co-transfected with sgRNA and Cas9 expressing vector and puromycin resistant
plasmid
(Mehravar et al., 2019)

10. RESULTS
1. RAG1 sgRNAs had better ability to induce Cas9-mediated introduction of DSBs at the RAG
locus and easy detection of RAG1 sgRNAs-induced indels by PCR. It was selected to
transfect into mES cells to target same 2 sites simultaneously.
2. CRISPR gene editing resulted in a multitude of engineered homozygous and compound
heterozygous mutations
- In-frame and out-of-frame indels in 92% of mES cell clones.
- Most of the mutations generated by CRISPR/Cas9 system were out-of-frame, resulting in
a complete gene knockout. In addition, 59% of the mutant ES cell clones carried out-of-
frame homozygous indel mutations.
- sgRNA-Cas9 Guided Genome Editing in RAG Genes.
1. RAG1 Knockout mES Clones
- Retained normal morphology & pluripotent gene expression.
1. Real-time PCR Results for Pluripotency Genes
- 3 Key markers for pluripotent stem cell (Oct4, Sox2, Nanog) revealed RAG1 knockout mES
cell line all (+ve) results.
(Mehravar et al., 2019)

11. DISCUSSION
Problems faced:
Mosaicism in embryos and off-target mutations.
Proposed action:
Utilise multiple different techniques and use a combined strategy
to avoid mosaic mutations.
OR produce gene edited embryos using CRISPR system via
mutant ES cells. (Has advantages over other techniques like
microinjection)
Rate of off-target editing can be reduced with properly
designed sgRNAs.

12. CONCLUSION
High-efficiency editing by CRISPR-Cas system can be achieved in mouse-cell line genomes at
targeted locations with efficient and well-targeted sgRNAs.
CRISPR/Cas9 system can efficiently create biallelic indels containing both homozygous and compound
heterozygous RAG1 mutations in about 92% of the mutant mESC clones. The 59% of mutant ES cell
clones carried out-of-frame homozygous indel mutations.
Genome editing results indicated that CRISPR/Cas9 system with correctly-designed sgRNAs generates
mutations in the desired genes and significant deletions can be achieved in the large number of cells
using two exonic gRNAs targeting one gene.
Faster generation of RAG1 knockout mice by creating chimera.
(Mehravar et al., 2019)

13. REFERENCES
Anders, C, Niewoehner, O, Duerst, A & Jinek M, ‘Structural basis of PAM-dependent target DNA recognition
by the Cas9 endonuclease’, Nature, Volume 513 (7519), pp. 569-573,
<https://www.ncbi.nlm.nih.gov/pubmed/25079318>.
Bayat, H, Omidi, M & Sabri, S, 2016, ‘The CRISPR growth spurt: from bench to clinic on versatile small RNAs’,
Journal of Microbiology and Biotechnology, volume 27 (2), pp. 207-218,
<https://www.researchgate.net/publication/310316039_The_CRISPR_growth_spurt_from_bench_to_clinic_on_v
ersatile_small_RNAs>.
Mehravar, M, Shirazi, A, Mehrazar, MM, Nazari, M, Banan, M & Salimi, M, 2019, ‘Efficient Production of Biallelic
RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9’, Iranian Journal of Biotechnology,
Volume 17(1), pp. 45-53, <http://www.ijbiotech.com/article_75677_fe57b228f02583b5d37447986f9874aa.pdf>.
National Institutes of Health, 2019, ‘What are genome editing and CRISPR-Cas9?’, accessed 16 October 2019,
<https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting>.
Roth DB. 2014. V(D)J Recombination: Mechanism, errors, and fidelity. Microbiol Spectrum 2(6):MDNA3-0041-
2014.
Sternberg, SH, Redding, S, Jinek, M, Greene, EC & Doudna, JA, 2014, ‘DNA interrogation by the CRISPR RNA-
guided endonuclease, Nature, Volume 507 (7490), pp. 62-67.