Determining the Efficacy of CRISPR-Cas9 Mechanisms on the uppP Gene of Klebsiella pneumonia using Lysozyme Assay

Mohd Nasharudin Razak; Raha Abdul Rahim; Abdul Rahman Omar; Zunita Zakaria; Nurulfiza Mat Isa

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Determining the Efficacy of CRISPR-Cas9 Mechanisms on the uppP Gene of Klebsiella pneumonia using Lysozyme Assay

Author: Mohd Nasharudin Razak ¹ ; Raha Abdul Rahim ^{1
,

2
,

3} ; Abdul Rahman Omar ^{2
,

4} ; Zunita Zakaria ^{2
,

4} ; Nurulfiza Mat Isa ^{1
,

2}
Author Information

1. Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia&
2. Institute of Biosciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia&
3. Chancellory, Universiti Teknikal Malaysia, 76100, Durian Tunggal, Melaka, Malaysia
4. Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
Publication Type:Journal Article
Keywords: CRISPR-Cas9, ESBL, Klebsiella pneumoniae, uppP gene, Cell growth and protection
From:Malaysian Journal of Medicine and Health Sciences 2020;16(No.4):226-233
CountryMalaysia
Language:English
Abstract: Introduction: Multidrug resistance bacteria is alarming worldwide. A lot of research were done and are ongoing to search for the best, convenient and economically affordable ways to fight them. With the latest genome editing tool; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, this research was performed to develop a novel strategy to genetically modify the genome and inhibit the growth of Klebsiella pneumoniae (UPM ESBLKP1), an Extended Spectrum Beta Lactamases (ESBL) organism. Methods: A CRISPR-Cas9 vector was constructed together with guide RNAs designed specifically for the targeted uppP gene, a gene responsible for bacterial cell growth and protection. Results: The growth and cell wall integrity of the modified Klebsiella pneumoniae (ΔUPM ESBLKP1) were significantly inhibited and reduced, respectively. Interestingly, wild type Klebsiella pneumoniae showed a normal growth curve while modified strains showed a faster doubling rate when supplemented with Luria-Bertani media. In contrast, slower growth rate of modified strain was observed in the M9 minimal media. This explained the higher doubling rate of mutants on nutrient rich medium earlier is being related to gene recovery. They grew slowly in the minimal media as they were adapting to a new environment while recovering the uppP gene and surviving, proving the success of its gene modification. Conclusion: The developed CRISPR-gRNA system was able to modify the targeted Klebsiella pneumoniae gene hence providing an opportunity to develop a new drug for Klebsiella pneumoniae infection as an alternative to antibiotics.
Full text:11.2020my0956.pdf