1. Genome editing by CRISPR
Yang De Marinis
PhD, Assistant Professor
Diabetes and Endocrinology
Lund University Diabtes Centre

2. Discovery of CRISPR

3. CRISPR identified

4. CRISPR identified

5. CRISPR identified
CRISPR

6. Immune system against virus
CRISPR in bacteria-
Immune system against virus
RNA harboring the spacer sequence helps Cas proteins recognize and cut exogenous DNA

7. CRISPR/Cas9 genome editing tool
The legend of three kingdoms
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes.
Jennifer Doudna
Emmanuelle Charpentier
Feng Zhang

8. CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.

9. Genome editing by CRISPR
Cleaved, expressed and processed. Forming CRISPR/Cas complex, nuclease

10. CRISPR/Cas9 structure Protospacer adjacent motif (PAM, NGG)
Cleaved, expressed and processed. Forming CRISPR/Cas complex, nuclease
Protospacer adjacent motif (PAM, NGG)

11. CRISPR/Cas9 action
Cleaved, expressed and processed. Forming CRISPR/Cas complex, nuclease

12. CRISPR mutations
CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.

13. CRISPR/Cas9 applications
Cleaved, expressed and processed. Forming CRISPR/Cas complex, nuclease
Gene Insertion
Gene Disruption
Gene Correction

14. CRISPR workflow 20-30% Western blot qPCR
CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.
Western blot
qPCR

15. An example of CRISPR application
3-4 bps from the PAM seq
PAM seq
PAM seq
3-4 bps from the PAM seq
P. Bompada et al., Int J Biochem Cellbiology, 2016 Oct

16. Target deletion of Ep300 exon1 by CRISPR in INS1 832/13
CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.

17. Target deletion of Ep300 exon1 by CRISPR in INS1 832/13
CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.

18. Histone modification: acetylation, methylation…
Reduced H3K9ac enrichment at Txnip gene promoter in CRISPR-ko Ep300 cell line
HDAC
CRISPR
p300 A A
ChREBP
ChREBP H3
TXNIP H3
Histone modification: acetylation, methylation…
turns a gene ”on” or ”off”
Activation: H3 acetylation, H3K4me3, H3K4me1
Repression: H3K27me3
Measurement of histone modification
Chromatin immunoprecipitation (ChIP)
Histones have highly positively charged tails, attracts negatively charged DNA tightly winds around the core octomer. Acetylation and methylation can neutralize the positive charge, loose up the chromatin structure, expose binding sites to transcription factors and transcription can be initiated.
Two of each of the core histones assemble to form one octameric nucleosome core, 147 base pairs of DNA wrap around this core particle. All histones have a highly positively charged N-terminus with many lysine and arginine residues. Addition of an acetyl group has a major chemical effect on lysine as it neutralises the positive charge. This reduces electrostatic attraction between the histone and the negatively charged DNA backbone, loosening the chromatin structure; Histone deacetylases remove those acetyl groups, increasing the positive charge of histone tails and encouraging high-affinity binding between the histones and DNA backbone. The increased DNA binding condenses DNA structure, preventing transcription.
E-box1
E-box2
- Reduced Txnip gene expression
- Reduced glucose-induced cell death
- Increased insulin secretion

19. ChIP seq on genome-wide H3K9ac enrichment on CRISPR-ko Ep300 cell line
We have optimized ChIP seq protocol by a pilot study performed on a beta cell line which has histone acetyltransferase p300 mutation introduced by CRISPR/Cas9. All procedures of ChIP seq have been optimized and data is currently under final statistic analysis.
Sequencing

20. CRISPR/Cas9 applications
Cleaved, expressed and processed. Forming CRISPR/Cas complex, nuclease
Gene Insertion
Gene Disruption
Gene Correction

21. Genom edit by CRISPR in humans
Human mutations
•Evolution
•Diseases
CRISPR solution
•Precise edit
•Editing at DNA level
Challenges
• Ethics
• Precision
CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.

22. Precise genome editing by CRISPR
CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.

23. CRISPR workflow 0.2% Western blot qPCR
CRISPR: specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks.
Western blot
qPCR

24. Genome edit by CRISPR No limit! • Seek (CRISPR gRNA) and cut (Cas9)
• Permanant edit at DNA level
• Edits: deletion, replacement, insertion
• Fast and low cost
• Limit:
No limit!

25. Learn to disign a CRISPR gRNA
Cleaved, expressed and processed. Forming CRISPR/Cas complex, nuclease