• 专利标题:   New engineered clustered regularly interspaced short palindromic repeats-associated 13 enzyme useful in composition for detecting presence of target nucleic acid e.g. viral nucleic acid in sample, has enhanced collateral activity.
  • 专利号:   WO2023102221-A1
  • 发明人:   GAO X, YANG J, GAO Y
  • 专利权人:   UNIV RICE WILLIAM MARSH
  • 国际专利分类:   C07K019/00, C12N009/22, C12N009/78, C12Q001/6816, C12Q001/6888
  • 专利详细信息:   WO2023102221-A1 08 Jun 2023 C12N-009/22 202352 Pages: 127 English
  • 申请详细信息:   WO2023102221-A1 WOUS051708 02 Dec 2022
  • 优先权号:   US285304P

▎ 摘  要

NOVELTY - An engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated 13 (Cas13) enzyme comprising a binding domain fusion, is new. The engineered Cas13 enzyme has enhanced collateral activity as compared to a Cas13 enzyme. USE - The engineered Cas13 enzyme is useful in composition for detecting presence of target nucleic acid in sample. The sample is a biological sample or environmental sample, preferably saliva, urine, plasma, or serum. The target nucleic acid is a viral nucleic acid or miRNA. The viral nucleic acid is viral RNA chosen from SARS-CoV-2 RNA, SARS-CoV-2 N gene, SARS-CoV-2 RdRp gene, Zika virus POLY gene, dengue virus POLY gene or Ebola virus L gene. The miRNA is hsa-miR-19b or hsa-miR-2392 (all claimed). The composition is also useful for typing bacterial strain, performing sensitive genotyping, and detecting disease-associated cell free DNA or RNA. ADVANTAGE - The engineered Cas13 enzyme has enhanced collateral activity, and ensures rapid and ultrasensitive detection of target nucleic acids at attomolar sensitivity. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the following: a method (A) for enhancing collateral activity of a Cas13 protein involves fusing the Cas13 protein to a binding domain, testing the collateral activity of the fusion protein, and identifying the fusion protein as having enhanced collateral activity if its collateral activity is increased as compared to a Cas13 enzyme; a fusion protein being made by the above-mentioned method (A); a composition comprising (i) the engineered Cas13 enzyme or the fusion protein and (ii) a CRISPR RNA (crRNA); and a method (B) for detecting presence of a target nucleic acid in a sample involves contacting the sample with the composition, and detecting a signal from the detectable label.