CRISPR-Cas Systems In Clinical Biochemistry: A New Frontier For Molecular Diagnostics

This study utilized experimental research design to contrast and evaluate diagnostic precision of CRISPR-Cas-based tests with established molecular diagnostic systems, i.e., polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Identification of specific nucleic acid targets from model clinical samples containing known viral RNA and bacterial DNA and identification of genetic mutations were the focuses of research. The study design allowed controlled assessment of sensitivity, specificity, turnaround time, and utilization of resources by the three diagnostic techniques. Clinical samples were spiked artificially with predetermined concentrations of target analytes. Samples were categorized into three groups: Group A (PCR-based detection), Group B (ELISA-based detection of protein targets), and Group C (CRISPR-Cas-based detection using Cas12 and Cas13 enzymes). The CRISPR assays used fluorescence-based reporter systems—SHERLOCK (Cas13a) and DETECTR (Cas12a)—and were carried out under isothermal conditions with the aid of recombinase polymerase amplification (RPA) for nucleic acid pre-amplification. All the experiments were carried out in triplicate, with appropriate positive and negative controls to determine validity as well as reproducibility of results. Variables measured for were: (1) sensitivity (expressed as the limit of detection in copies/µL), (2) specificity (ability to distinguish target from non-target sequences), (3) time to result (minutes), and (4) cost per test. Measurements were recorded with a fluorometer for the CRISPR tests, spectrophotometer for ELISA, and real-time thermal cycler for PCR. Data analysis was performed using SPSS version 27.0. Analysis of variance (ANOVA) and follow-up post hoc Tukey tests were used to find out significant differences (p < 0.05) in sensitivity, specificity, and time efficiency among the diagnostic platforms. All the laboratory experiments were conducted under biosafety level 2 (BSL-2) conditions. Reagents and CRISPR diagnostic kits were supplied by certified suppliers. The experiments were conducted under a well-equipped molecular diagnostics laboratory.