Degenerate sequence-based CRISPR diagnostic for Crimean –Congo hemorrhagic fever virus

by Hongzhao Li, Alexander Bello, Greg Smith, Dominic M. S. Kielich, James E. Strong, Bradley S. Pickering CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a “spacer” sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR fu nction can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target varia tions. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based S HERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean–Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic ...
Source: PLoS Neglected Tropical Diseases - Category: Tropical Medicine Authors: Source Type: research