Role of CRISPR-Cas in antimicrobial research

1. Course work seminar on the topic of “Role
of CRISPR-Cas9 in antimicrobial resistance”
By-
Anmol Kulshrestha
Ph.D.

2. TABLE OF CONTENT
 Introduction
 Components of CRISPR-Cas9 system
 Structure of Cas9 & sgRNA
 Mechanism of CRISPR-Cas9 system
 Advantages
 Limitations
 Application
 Role of CRISPR-Cas system in antimicrobial resistance

3. INTRODUCTION
• CRISPR-Cas9 is a unique technology that enables scientists to edit
parts of the genome by removing, adding or altering sections of the
DNA sequence.
• It was first observed in E.coli by Osaka University researcher
Yoshizumi Ishino in 1987.
• In 2012, Dr. Jennifer Doudna and Dr. Emmanuel Charpentier
revealed that the CRISPR-Cas9 bacterial immune system could be
repurposed as a gene editing tool.
• Provides adaptive immunity against viruses.

4. COMPONENTS OF CRISPR-CAS9 SYSTEM

5. STRUCTURE OF CAS9
Cas9
REC
REC I
REC II
NUC
HNH domain
RuvC domain
PAM domain

6. STRUCTURE OF SGRNA

8. MECHANISM OF
CRISPR-CAS9 SYSTEM

9. ADVANTAGES
More
accurate
Target
specific
Multi-
potential
Easy to
use

10. LIMITATIONS
Off-targets
Unavailability of
PAM
Immunogenicity In-vivo toxicity

11. APPLICATION
 Diagnosis; ex – FELUDA
 Antimicrobial resistance; ex – methicillin, kanamycin
 Genome sequencing; ex - identify genes associated with cancer cell
survival, drug resistance.
 Gene therapy; ex – infectious diseases and cancer
 Plant Biotechnology; ex – increasing yield, improving quality,
disease resistance

12. Role of CRISPR-Cas system in antimicrobial
resistance

13.  Therefore, the CRISPR-Cas system can be programmed to
specifically target and cleave any DNA for in vivo based on the
information provided in the CRISPR array, which has been
exploited to target the bacterial population which carry specific
genes that encode antibiotic resistance.
 First, the construction of a transferrable CRISPR-Cas system which
spacers that targets conserved sequence of the resistance genes.
 Introduction of this CRISPR-Cas system into a bacteriophage in
place of genes responsible for phage growth.
 Then the phage was used insert into the bacteria.

14.  In this way, due to high specificity of phages to their host, in
combination with the specificity of the CRISPR-Cas construct that
targets resistance-conferring strains complex bacterial populations.
 This feature may enable the targeting of the pathogens posing the
most relevant threats, with minimal disturbance of the ecological
niche of other microbes.

15. STUDIES HAVE BEEN DONE SO FAR
Target Resistant Test
microorganism
References
ndm-1 & ctx-m-15 carbapenem E. coli (Yosef et al. 2015)
aph-3 kanamycin S. aureus (Bikard et al. 2015)
tem ceftazidime E. coli (Kim et al. 2016)
ndm-1 & shv-1 Kanamycin E. coli (Citorik et al. 2014)
mecA Methicillin S. aureus (Kiga et al. 2020)

16. THANK YOU