2 results about "Selective cleavage" patented technology

This specification discloses a method for high-throughput evaluation of the in vitro cleavage activity of a CRISPR / Cas system. The inventors of this application have implemented this method based on the concept that an in vitro environment can be simulated by immobilizing cells to inhibit their viability, while partially utilizing existing established high-throughput evaluation methods for in vivo environmental activity. Furthermore, this specification discloses a model for predicting the cleavage activity of a CRISPR / Cas system and its application method. This includes: 1) collecting cleavage activity data using various combinations of target-guide vectors; 2) obtaining two or more alignment patterns to augment the data when the target nucleic acid and guide nucleic acid do not match; and 3) inventing the model by developing a method for training the model using aligned target nucleic acid sequences and aligned guide nucleic acid sequences as key input variables, and applying the model in various ways. Additionally, this specification discloses a method for enriching rare nucleic acids using a CRISPR / Cas system when a sample contains trace amounts of rare nucleic acids and a large amount of background nucleic acids. The inventors selected a guide nucleic acid (optimized guide nucleic acid) that promotes selective cleavage of the nucleic acid to be cleaved (rare nucleic acid or background nucleic acid) relative to nucleic acids with similar sequences (background nucleic acids or rare nucleic acids), and used it to develop a rare nucleic acid enrichment method.

This invention relates to the optimization and application of C1 type botulinum toxin light chain (BoNT / C1LC) mutants. Using the selectively Syntaxin-1 cleaving mutant BoNT / C1α-51 as a template, this invention employs PCR site-directed mutagenesis to replace amino acids at positions 53-51 and evaluates enzyme activity at the cellular level. The results show that mutants 53Y, 52H53Y, 51D52H53Y, 51S52H53Y, 51G52H53Y, 51A52H53Y, and 51Y52H53Y exhibit no cleavage activity against the substrate SNAP-25, but retain cleavage activity against Syntaxin-1. Among these, 52H53Y, 51S52H53Y, and 51G52H53Y show superior Syntaxin-1 cleavage activity compared to BoNT / C1α-51. The mutants provided by this invention, which replace full-length BoNT / C1LC or are fused with a membrane-penetrating peptide and are active only for Syntaxin-1, can be used in the preparation of medical aesthetic products and related drugs that supplement clinical responses to or non-responses to botulinum toxin type A, and whether or not resistance has been developed. Mutants with Syntaxin-1 cleavage activity superior to BoNT / C1α-51 can be used in the preparation of drugs for Syntaxin-1-mediated hypersecretion diseases. Mutants such as 51F52H53Y have no cleavage activity against both SNAP-25 and Syntaxin-1 and can be used in the preparation of targeted delivery drugs for the nervous system using full-length botulinum toxin as a carrier.