Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPR) is an array of short repeated DNA sequences interspaced with spacers having unique sequences, which has been found in approximately 50% of the bacterial genomes and in 87% of the genomes of archaea so far [1]
We introduce the various diagnostic methods based on the CRISPR-Cas system, focusing on the functional activity of Cas proteins used for detecting disease-related genetic molecules and the various form of the genetic molecules such as genomic DNA, mRNA, microRNA, single nucleotide polymorphism (SNP)
Nucleic acids are the main target of the CRISPR-Cas system, so various forms of nucleic acids such as genomic DNA, mRNA, miRNA, SNP, and methylated DNA
Summary
Clustered regularly interspaced short palindromic repeats (CRISPR) is an array of short repeated DNA sequences interspaced with spacers having unique sequences, which has been found in approximately 50% of the bacterial genomes and in 87% of the genomes of archaea so far [1] It was firstly discovered in the Escherichia coli genome in 1987 as repeated 29-nt fragments separated by 32-nt fragments, and the first evidence to show that the CRISPR-Cas system is a part of an adaptive immune system in archaea or bacteria was reported in 2005 [2,3]. Biomolecules 2021, 11, 1162 attention to CRISPR and CRISPR-based diagnostics are well reflected in the histograms displaying the number of the published research papers per year over the last few decades that were searched by PubMed (https://pubmed.ncbi.nlm.nih.gov, accessed on 10 May. 2021) using one keyword, “CRISPR” (Figure 1C, left) and two keywords “CRISPR” and “diagnosis” (Figure 1C, right). CRISPR-based diagnostic methods for detecting non-nucleic acids and non-amplified targets are discussed briefly in the last part of this review
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