**3.4 CRISPR-Cas-based analysis of nucleic acids**

CRISPR-Cas is known to endow bacteria and archaea adaptive immunity against foreign- nucleic acids using mobile genetic elements [85]. CRISPR-Cas proteins cleave invading DNA- and generate spacer nucleotides known as protospacer. The protospacer integrates into- genome near the protospacer adjacent motif (PAM) region required as memory for future- interrogation and cleavage of same invader depending on spacer-phage similarity. For the targeting, CRISPR-Cas9 requires a gRNA (guide RNA) composed of *tracr*RNA (trans-activating)- and crRNA, or a chimeric sgRNA (single guide RNA). The RNA-guided cleavage is mediated- by RuvC (a member of RNase H family) and HNH catalytic domains at the site of gRNA-target sequence base-pairing. In this mechanism, Cas9-gRNA complex recognizes a G-rich PAM- region of the target sequence followed by blunt end cleavage. However, some Cas enzymes- such as Cas9 and Cas12a, purified from *Francisella tularensis novicida* and *Streptococcus pyogenes,* exhibit nonspecific RNA-independent DNA cleavage in the presence of Mn2<sup>+</sup> [86], suggesting the significant role of several mediators including Cas RuvC nuclease domain. On the- basis of gRNAs, more literatures indicated that Cas12a, Cas13a, and Cas13b enzyme effectors- require a mature crRNA for self-assembly and processing and ribonucleoprotein surveillancedependent nuclease or DNA for interference activity [87, 88]. Moreover, the Cas12a, Cas13a,- and Cas13b enzymes do not require a dual functional crRNA-TrancRNA as for Cas9 [89, 90].-

Basically, CRISPR-Cas systems are categorized into three main types (type I, type II, and- type III) and 12 subclasses based on the genetic and structural differences [91]. From these- classes, Type II CRISPR-Cas is widely used for genome-editing applications. Currently,- researchers have exploited the CRISPR-Cas system in diagnostics. Two CRISPR-Cas-based- diagnostic systems termed DETECTR (DNA Endonuclease Targeted CRISPR *Trans* Reporter)- and SHERLOCK (Shorthand for Specific High Sensitivity Reporter unLOCKing) have been- developed as a new platform for real time detection of nucleic acid based on Type II Cas13a- and Cas12a, respectively. These enzymes exhibit collateral cleavage of nucleic acid targets- and nontarget single strands in vicinity. For instance, Cas13, an RNA-targeting CRISPRassociated type VI-A protein, cleaves an ssRNA at a non-G PFS (protospacer flanking site)- of target sequence in a gRNA-independent manner via its endonuclease HPN domain [12]. On the contrary of Cas13a and distinct from Cas9, Cas12a (cpf1) is a CRISPR-Cas family enzyme that possess a unique RNA-guided DNase activity [88]. It targets at 5' T-rich- PAM region (TTTN) by cleaving ~18 nucleotides on the DNA strand opposite to the gRNA- complementary strand by leaving 5 nucleotide staggered cuts on both 5′-ends of the target- sequence [92]. Cas12a can collaterally cleave both targeted dsDNA and a nontarget ssDNA- in vicinity. Through the integration of a fluorescently labeled ssDNA reporter, a detectable- signal can be obtained after cleavage. However, the cleavage of reporter nucleic acids is- motif dependent and requires 41–44 nucleotide crRNA to recognize a 5′ T-rich PAM of the- target sequence, while Cas9 requires ~100 nucleotide gRNA at the 3'G-rich PAM target site.- Nevertheless, Cas12a is unable to *trans-*cleave an ssRNA reporter and a targeted ssRNA- sequence [11], suggesting that it exhibits only a DNA-activated DNase activity. In contrast,- Cas13 enzymes (e.g., CcaCas13b and LwaCas13b) from some bacteria strains have shown a- random enriched motif cleavage with U-dependent nucleotide preference, while some other- Cas13 enzymes (e.g., PsmCas13b and AsCas12a) strongly preferred A-nucleotides and A-T- dinucleotide across the motif, respectively [11]. Nevertheless, other CRISPR-Cas13a/b exhibited dinucleotide preference collateral cleavage activity. This activity can be enhanced with- optimized target concentration, buffer, and crRNAs. Therefore, irrespective of the targetefficiency of the CRISPR/Cas system, it is consent that a single-guided RNA-Cas enzyme- complex recognition of target nucleic acid and reaction conditions is required to initiate- cleavage of both target and a nearby nontarget (reporter sequence).-

Various diagnostic applications necessitate detection of one or more targets, and therefore with- tremendous propensity of both platforms, CRISPR-Cas enzymes can detect a single or multiple- targets in complex liquid biopsy samples. Various samples suspected with Zika, dengue, and- human papilloma viruses and bacteria as well as SNP and mutation discrimination have been- developed. Their multiplex detection relies on reprogrammable crRNA tailing specific target- sequence and enriched multiple motif fluorescent reporters. For example, Gootenberg etal.- showed that isothermally amplified four different target nucleic acids could be detected simultaneously by LwaCas13a, PsmCas13b, CcaCas13b, and AsCas12a, with leveraged dinucleotide- motifs harboring FAM, TEX, Cy5, and HEX quenched fluorescent reporters, respectively. After,- the reporter is cleaved by Cas enzymes; the read-out can be achieved by high specific detection- of four different quenched fluorescent reporters or using lateral flow biosensor analysis with- specific antibodies against fluorescein-biotin reporters at conjugate pad and protein A as second- antibody immobilized at the control line (**Figure 3**). Thus, these enzymes are intriguing for broad- spectrum diagnostic applications (**Figure 3,** left). Moreover, the combination of isothermal amplification and CRISPR-Cas system for amplification and signal readout, respectively, revealed an- amplified signal detection of 8zM in a 250μl reaction volume. It should be noted that isothermal- preamplification of target nucleic acids is crucial to achieve that robust sensitivity with Cas12- and Cas13 enzymes [13]. This approach could be adopted, however with the most minimized- cost. More interestingly, it is simple, fast, specific, sensitive, and can be multiplexed. Thus, it is- convenient in minimally instrumented fields for point-of-care detection of nucleic acids.-

**Figure 3.** Workflow of nucleic acid detection with the CRISPR-Cas system**.** Nucleic acids are obtained from samples by proteinase K treatment or heat treatment. The nucleic acids are isothermally amplified using recombinase polymerase amplification at 37°C for 10min. A one-pot reaction comprising amplicons, Cas protein, a designed crRNA for specific DNA/RNA target spotting, and reporters is prepared. To detect the presence of target nucleic acid, a fluorophore molecule (yellow star) and a quenching molecule (circle) are used. When Cas protein slices its nucleic acid target, and any ssDNAreporter nearby, the quenching molecule frees from the fluorophore, letting it fluoresce. The fluorescence can be detected directly or the reaction mixture can be applied to lateral flow assay.-
