Surface-Enhanced Raman Spectroscopy for Monitoring the Biochemical Changes Due to DNA Mutations Induced by CRISPR-Cas9 Genome Editing in the Aspergillus niger Fungus.

Abstract

In this study, surface-enhanced Raman spectroscopy (SERS) technique, along with principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA), is used as a simple, quick, and cost-effective analysis method for identifying biochemical changes occurring due to induced mutations in the Aspergillus niger fungus strain. The goal of this study is to identify the biochemical changes in the mutated fungal cells (cell mass) as compared to the control/nonmutated cells. Furthermore, multivariate data analysis tools, including PCA and PLS-DA, are used to further confirm the differentiating SERS spectral features among fungal samples. The mutations are caused in A. niger by the clustered regularly interspaced palindromic repeat CRISPR-Cas9 genomic editing method to improve their biotechnological potential for the production of cellulase enzyme. SERS was employed to detect the changes in the cells of mutated A. niger fungal strains, including one mutant producing low levels of an enzyme and another mutant producing high levels of the enzyme as a result of mutation as compared with an unmutated fungal strain as a control sample. The distinctive features of SERS corresponding to nucleic acids and proteins appear at 546, 622, 655, 738, 802, 835, 959, 1025, 1157, 1245, 1331, 1398, and 1469 cm-1. Furthermore, PLS-DA is used to confirm the 89% accuracy, 87.7% precision, 87% sensitivity, and 88.9% specificity of this method, and the value of the area under the curve (AUROC) is 0.67. It has been shown that surface-enhanced Raman spectroscopy is an effective method for identifying and differentiating biochemical changes in genome-modified fungal samples.