CRISPR-CAS9 & DNA FINGER PRINTING

CRISPR-CAS9 & DNA FINGER PRINTING Umair Masood Awan Beyond its revolutionary role in genetic engineering, CRISPR-Cas9 is forging a new path in forensic science through a highly specific DNA detection system. This application leverages the system's core function: the guide RNA's ability to lead the Cas9 protein to a precise DNA sequence. In a forensic context, we can design a guide RNA that is complementary to a specific Short Tandem Repeat (STR) marker, a DNA sequence commonly used for identification. This guide RNA is also labeled with a fluorescent tag. When this complex is applied to a DNA sample, a crucial binary result occurs. If the sample lacks the exact matching STR sequence, the guide RNA and Cas9 cannot bind, and thus, no fluorescent signal is emitted under UV light. However, if the sample contains the target STR, the complex binds securely, and a distinct fluorescent signal becomes visible. This provides a clear, visual confirmation of the presence of that specific genetic marker, offering a potentially faster and more targeted alternative to traditional DNA profiling techniques. Fig: 1.1 CRISPR Cas9 Working Protocol:  Position a 30 µl reaction along micro centrifuge tube on ice with the following sequence.  Softly mix the reaction mixture and centrifuge it.  Incubate at 37°C for 20min. Table: 1.1 Results: Based on the observed UV light signals, the DNA samples from A, B, C, and H were successfully cleaved by the Cas9 protein. This indicates that the guide RNA correctly bound to complementary target sequences within these DNA samples, enabling the Cas9 complex to recognize and cut the DNA, resulting in the release of a fluorescent reporter. In contrast, the lack of signal from samples E, G, and F suggests that the guide RNA could not bind effectively to these DNA sequences. Fig: 1.2 Application DNA fingerprinting and CRISPR-CAS9 is used to:  Identify individuals.  Determine Genetic relationships.  Analyze Genetic Variation & similarities among Populations. References 1. Mnookin J L, Cole S A, Dror I E, Fisher B A (2010) the need for a research culture in the forensic sciences. UCLA L Rev 58:725. 2. Chambers GK, Curtis C, Millar CD, Huynen L, Lambert DM (2014) DNA fingerprinting in zoology: past, present, future. Invest Gen 5: 1. 3. Kimura P, Nakane T, Ishitani R, Hatada I, Zhang F et al. (2014) Molecular mechanism of CRISPR. J Appl Crystallogr 156: 935-949 4. Amann R, Ludwig W (2000) Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology. FEMS Microbio Rev 24: 555565.