National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, Canada.
National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada.
PLoS Negl Trop Dis. 2022 Mar 10;16(3):e0010285. doi: 10.1371/journal.pntd.0010285. eCollection 2022 Mar.
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 function 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 variations. 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 SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/μl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens.
CRISPR（成簇规律间隔短回文重复序列）是原核生物用来切割入侵病毒和质粒核酸的古老防御机制，目前正被世界各地的研究人员用于开发新的即时诊断方法。在 CRISPR 诊断中，含有与靶核酸序列特异性互补“间隔序列”的 CRISPR RNA（crRNA）指导 CRISPR 效应蛋白（Cas13a、Cas12a 或 Cas12b）的激活，导致 RNA 或 DNA 报告分子的旁切，从而实现巨大的信号放大。根据之前的研究，间隔序列与靶序列之间的某些类型的序列错配会破坏 CRISPR 的功能。这在检测 RNA 病毒等具有高度序列多样性的可变靶标时带来了潜在挑战，因为靶标变异会导致错配。为了覆盖病毒多样性，我们在本研究中提出，在 crRNA 合成过程中，可以在对应病毒序列变异的间隔序列位置引入混合核苷酸类型（简并序列）。我们在基于 Cas13a 的 SHERLOCK（特异性高灵敏度酶报告物解锁）技术背景下测试了这种 crRNA 设计策略，用于检测克里米亚-刚果出血热病毒（CCHFV），这是一种生物安全 4 级病原体，分布广泛，序列变异广泛。这种简并序列的 CRISPR 诊断具有功能性、敏感性、特异性和快速性。它可以在 30-40 分钟内检测到来自代表所有谱系的 CCHFV 毒株的 1 拷贝/μl 病毒 RNA，也可以检测到最近在 CRISPR 靶区出现新突变的新毒株。同样重要的是，它与各种 CCHFV 相关病毒没有交叉反应。这项概念验证研究表明，基于简并序列的 CRISPR 诊断是一种很有前途的选择，可用于有效检测高度变异的病毒病原体。
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