Development of CRISPR-Cas9-mediated site-directed mutagenesis of ECE1 gene (candidalysin) in Candida albicans

Engku Nasrullah Satiman, Engku Anis Fariha and Kaderi, Mohd Arifin and Ramzi, Ahmad Bazli and Arzmi, Mohd Hafiz (2020) Development of CRISPR-Cas9-mediated site-directed mutagenesis of ECE1 gene (candidalysin) in Candida albicans. In: Asian Regional Conference on Systems Biology 2020 (ARCSB 2020), 2nd-4th March 2020, Langkawi, Kedah. (Unpublished)

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Abstract

CRISPR-Cas9 has been extensively used in microbial editing considering its specificity and versatility. Cumbersome editing in C. albicans due to its aberrant chromosomal ploidy and lock of meiosis can be potentially solved using CRISPR-Cas9. In this study, the third KR (lysine-arginine) motif on the ECEI gene that encodes for candidalysin in C.olbicans is targeted. The third KR motif (AAGAGA) is substituted into KA motif (AAGGCA) through transformation of CRISPR-Cas9 ribonucleoprotein (RNP) complex, ssDNA and pYM70 that confers hygromycin B resistance into competent cells of C. albicans using electroporation. The cleaving efficiency of the designed sgRNA is tested using in vitro nuclease assay against several concentrations of RNP prior transformation. The RNP complex that formed through co-incubation of sgRNA and purified Cas9 induced double-stranded break to the nearest protospacer adjacent motif to the third KR site. The DNA break is repaired by co-transformed ssDNA that contains KA motif at the third KR nucleotides flanked by 90 bp homologous arms via homologous recombination. The transformants were grown on YPD infused hygromycin B (600 µg/ml) for growth and selection. Mutation in successfully grown colonies will be verified through sequencing. The designed sgRNA shows in vitro efficient cutting at RNP concentrations of 15 nM, 20 nM and 25 nM. CRISPR-Cas9 is expected to direct correct substitution of bases that encodes for alanine at the third KR motif of the ECE1 gene. This study shall contribute to new approaches of gene editing and site-directed mutagenesis in C. albicons as well as in other in vitro and in vivo models.

Item Type:	Conference or Workshop Item (Poster)
Additional Information:	4643/80883
Uncontrolled Keywords:	CRISPR-Cas9; candidalysin; Candida albicans
Subjects:	Q Science > QR Microbiology R Medicine > RK Dentistry
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button):	Kulliyyah of Allied Health Sciences Kulliyyah of Allied Health Sciences > Department of Biomedical Science (Effective:1st July 2011) Kulliyyah of Dentistry Kulliyyah of Dentistry > Department of Fundamental Dental and Medical Sciences
Depositing User:	AP Ts Dr Mohd Hafiz Arzmi
Date Deposited:	25 Jun 2020 14:32
Last Modified:	08 Jun 2022 14:15
URI:	http://irep.iium.edu.my/id/eprint/80883

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