pre_phd_ppt

Views:
 
Category: Sports
     
 

Presentation Description

Gene disruption by Cell penetrating peptide mediated delivery of cas 9 protein and guide RNA

Comments

Presentation Transcript

Slide1:

Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA Suresh Ramakrishna,1 Abu-Bonsrah Kwaku Dad,1 Jagadish Beloor,2 Ramu Gopalappa,1 Sang-Kyung Lee,2 and Hyongbum Kim1,3 1Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea; 2Department of Bioengineering and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul 133-791, Republic of Korea Presented by: Rajesh Supervisor: Dr. G P S Raghava

Introduction to CRISPR - Cas:

Introduction to CRISPR - Cas CRISPR – Clustered, regularly interspaced, short palindromic repeats. CRISPR-Cas system targets DNA or RNA as a way of protecting against viruses and other mobile genetic elements. The CRISPR locus, first observed in Escherichia coli is present in about 84% of archaea and 45% of bacteria according to the most recent update of the CRISPRdb. Cas – CRISPR – associated Three types of CRISPR mechanisms have been identified, of which type II is the most studied. Science 327 (5962): 167–70

Mechanism of CRISPR-Cas :

Source: CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology Mechanism of CRISPR- C as

Engineered CRISPR-Cas system :

Source: CRISPR-Cas systems for editing, regulating and targeting genomes ( Nature review) Engineered CRISPR- Cas system

Hypothesis of the study:

Efficient genome editing using CRISPR - Cas requires the successful delivery of this system in to cells. Plasmid mediated delivery, microinjection, electroporation etc. Plasmid based delivery also has some other complications – Uncontrolled integration of plasmid DNA into host genome. Unwanted immune response caused by bacterial sequences ( Hemmi et al. 2000 ; Wagner 2001 ). Safety problem if antibiotic resistance gene is taken up by pathogenic bacteria. Hypothesis of the study So, a novel method is required which can deliver CRISPR- cas genome editing system directly into cells, without additional tools. So, proposed hypothesis is the use of Cell penetrating peptide for the efficient delivery of CRISPR- cas .

Scheme of study:

Expression and purification of cas 9 protein Scheme of study In-vitro cleavage assay to check activity of cas 9 (cas9-m9R) Conjugation of cas 9 with CPP sgRNA preparation and complexing with CPP (sgRNA:9R) Cell based reporter assay to monitor cas9-m9R and sgRNA:9R function Endogenous gene disruption by cas9-m9R and sgRNA:9R Analysis of genome editing with various cell lines and advantage of CPP based method over other methods

Expression and purification of cas 9 protein :

Expression and purification of cas 9 protein Expression of His-tagged Cas9 protein in BL21 E. coli cells. Cas 9 protein in E.coli cell was expressed and purified by Ni-NTA column

In vitro DNA cleavage by the purified Cas9 protein :

In vitro DNA cleavage by the purified Cas9 protein In vitro DNA cleavage by the purified Cas9 protein. Cas9 protein cleaves target DNA only in presence of sgRNA .

Conjugation of CPP to cas9 protein:

Conjugation of CPP to cas9 protein Mass spectrometry of the Cas9 and Cas9-m9R Cas9 = 167.3KDa Cas9-cys = 167.4KDa Cas9-cys-m9R = 169.4KDa Mass spectrometry showed that a suitable fraction of cas9 protein was conjugated with m9R.

sgRNA preparation and complexing with CPP:

sgRNA preparation and complexing with CPP 9R and sgRNA form condensed , positively charged nanoparticles which possibly facilitates delivery of sgRNA into cells. A. Electrophoretic mobility shift assays B. Et:Br size exclusion assay C. Dynamic light scattering D. Zeta potential E. SEM

Reporter assay to monitor cas9-m9R and sgRNA:9R function:

Reporter assay to monitor cas9-m9R and sgRNA:9R function Since GFP expression was not observed in negative controls, so this data suggests that cas9-m9R and sgRNA:9R can enter human cells and cleave target DNA sequences.

Endogenous gene disruption by cas9-m9R and sgRNA:9R:

Endogenous gene disruption by cas9-m9R and sgRNA:9R Simultaneous treatment of cells with cas9-m9R and sgRNA:9R improve the genome editing effieciency .

Reduced off-target cleavage effects of cas9-m9R and sgRNA:9R:

Reduced off-target cleavage effects of cas9-m9R and sgRNA:9R The off-target mutation frequencies in cells treated with Cas9-m9R and sgRNA:9R were drastically lower than those treated with plasmids, whereas the on- target mutation frequencies were comparable between the two groups.

Factor that affect the genome editing efficiency of Cas9-m9R and sgRNA:9R:

Factor that affect the genome editing efficiency of Cas9-m9R and sgRNA:9R

Application of Cas9-m9R and sgRNA:9R to several human cell types:

Application of Cas9-m9R and sgRNA:9R to several human cell types HEK293T- Human embryonic kidney cells HeLa - Human cervical cancer cell line NCCIT -Human embryonal carcinoma cell line HDF -Human dermal fibroblast H9 -Human embryonic stem cell

Conclusion:

CPP can enable delivery of cas9 protein and sgRNA into cultured mammalian cells and also cause endogenous gene disruptions. CPP based delivery also circumvents potential problem associated with plasmid based methods. A cloning free process which can readily be customized for genome editing at loci of interest. CPP mediated delivery reduces off target effects relative to plasmid mediated delivery. CPP based approach might represent a major step toward potential development of “ in vivo genome editing ” which can be potentially applicable for human patients. Conclusion

Slide17:

Thank you

authorStream Live Help