| Abstract [eng] |
CRISPR (clustered regularly interspaced palindromic repeats) systems are sought-after genome editing tools for therapeutics and diagnostics. The use of CRISPR systems for gene editing is not without difficulties: the specificity of Cas12a nuclease, a Cas9 alternative, presents a limit in its use for gene editing due to its inability to successfully edit some genes. The primary cause of this problem is believed to be gRNA underperformance, caused specifically by gRNA secondary structures. The gRNA plays a critical role in CRISPR systems. Experimental analysis has shown that the use of CRISPR for genome editing can result in off-targeting, causing a high degree non-specific editing at undesired genomic. Strategies used for gRNA evaluation have several drawbacks as well, such as a need for substantial protein engineering, incompatibility with viral packaging restrictions, and an increased number of system components. Published findings on the impact of the nucleotide composition and secondary structure of the gRNA contradict one another. Although, guides with slight modifications like changing the nucleotide composition and extension of the secondary structures can efficiently improve Cas12a nuclease efficiency for gene editing . Several bioinformatic tools help users design guide RNA with high targeting capability. Yet, the effectiveness of gRNAs in guiding Cas12a for genome editing varies substantially. Therefore, the field needs methods for designing highly effective gRNAs that can improve target identification and reduce nonspecific targeting. For this research, 12,000 gRNAs, spanning different nucleotide compositions, targets and secondary structures will be analyzed to show how these features influence transcription, ribonucleoprotein complex formation, and target cleavage. We will apply high-throughput next- generation sequencing to characterize gRNAs at each stage. This study's logical design of gRNAs is a viable way to meet this demand. Using well- characterized oncogene target locations, we will show that rationally designed RNA secondary structures can boost the efficacy and specificity of gene editing tools like Cas 12a. |
