The CRISPR/Cas9 system is composed of two main components: the Cas9 protein and the guide RNA (gRNA) complex, which includes gRNA consisting of trans-activating CRISPR RNA (tracrRNA) and crRNA. This system has been successfully implemented in a wide range of model bacteria, such as E. coli, Mycobacterium spp., Lactobacillus spp., and Streptomyces spp. [121]. When pathogen DNA invades bacteria, the Cas9 system first synthesizes a gRNA that incorporates an RNA sequence that matches the target site. When DNA binds to the gRNA complex, it is degraded by the Cas9 protein. Fig. 3 illustrates how Cas9 binds to the target nucleic acid and activates the corresponding signal molecules. Nucleic acid detection using the Cas9 system depends on this characteristic. A unique gRNA is designed according to the target DNA and can thus detect different genes [122].

Many detection platforms for CRISPR/Cas9 have been developed and utilize its cleavage and endonuclease (NEase) activity. Nucleic Acid Sequence-Based Amplification-CRISPR Cleavage (NASBACC) successfully differentiated between Zika virus (ZIKV) strains in America and Africa using the PAM region in the target sequence for Cas9. Through Nucleic Acid Sequence-Based Amplification (NASBA) amplification, the sensor detected the amplicon of the American ZIKV, which includes the PAM region and was cleaved by Cas9, while the African ZIKV, which lacks the PAM region, was not cleaved [54].

Attempts to treat Human Immunodeficiency Virus-1 (HIV-1) with CRISPR/Cas9-based therapies were first made in 2013. CRISPR/Cas9 could inhibit the expression of HIV-1 genes by targeting the HIV-1 long terminal repeats (LTR) [123]. The ability of CRISPR to eliminate integrated viral genes within the infected host chromosome implies that Cas9 could present a promising approach for addressing AIDS [124]. In addition, a colorimetric technique for identifying viruses using the CRISPR/Cas9 system was reported in 2020 [125]. This approach involves directly detecting RNA from the viral lysate using the CRISPR/dCas9 system combined with a biotin PAM presenting oligonucleotide (PAMmer). Under the guidance of biotin-PAMmer, gRNA can recognize RNA in viral lysates, and streptavidin-horseradish peroxidase (HRP) and biotin-PAMmer react to induce color changes. SARS-CoV-2, drug-resistant influenza A virus (pH1N1/H275Y), and influenza A virus (pH1N1) were successfully identified by observation with the naked eye [125].

The CRISPR/Cas9-isothermal exponential amplification reaction (CAS-EXPAR) method is based on isothermal amplification utilizing CRISPR/Cas9 technology. PAM presents oligonucleotides to activate Cas9-specific cleavage activity for product cleavage, and then EXPAR amplifies and cleaves the product. The amplification product is combined with fluorescent dye to produce fluorescence. The CAS-EXPAR detection technology is a highly adaptable method for identifying various types of nucleic acids, including DNA, RNA, and methylated DNA, and it has been successfully used to detect DNA methylation and total RNA from L. monocytogenes [55]. Integration of CRISPR/Cas9 technology with methylation sequencing techniques offers a powerful tool for studying DNA methylation patterns and their functional significance. CRISPR/Cas systems can be engineered to target specific genomic loci and introduce DNA methylation changes. This can be achieved by fusing catalytically inactive Cas proteins (dCas) with DNA methyltransferase domains or recruiting endogenous DNA methyltransferases to targeted loci using gRNAs. By precisely manipulating DNA methylation levels at specific genomic regions, researchers can investigate the functional consequences of methylation changes on gene expression, chromatin structure, and cellular phenotype [126].

In 2019, a nucleic acid detection platform called “finding low abundance sequences by hybridization” (FLASH) was established. This platform combines Cas9 cleavage, PCR amplification, and high-throughput NGS [24]. The gRNA was specifically designed to direct the Cas9 nuclease to a targeted gene sequence for cleavage. The spliced sequences were amplified by PCR and then sequenced by high-throughput sequencing. FLASH-NGS could effectively detect antimicrobial resistance genes in various samples in a highly multiplexed manner (Fig. 4C).

Another important approach of CRISPR/Cas9 for methylation sequencing is targeted methylation sequencing, especially using nanopore technologies. Unlike most other sequencing methods that require amplification of the target gene, which can lead to amplification bias, or require nucleic acid conversion as in bisulfite sequencing approaches, nanopore sequencing technology can directly identify various base methylations bioinformatically. However, these non-amplifying methods require large amounts of DNA to ensure sufficient genome coverage [127–130]. Many studies have showcased the effectiveness of combining CRISPR/Cas9 with long-read sequencing for nanopore analysis. The application of the CRISPR/Cas9 platform has demonstrated promising results in achieving the enhanced sequence coverage necessary for the precise identification of genetic variations. This breakthrough has facilitated the discovery of distinct methylation patterns between afflicted and unaffected individuals, leading to the detection of novel hypomethylated regions within the genome. For instance, Gilpatrick and colleagues developed an innovative approach: nanopore Cas9-targeted sequencing (nCATS) that eliminates the need for amplification by using the CRISPR-Cas9 system to target and cleave a specific region of the genome, and the enriched fragments are subjected to long-read nanopore sequencing [127] (Fig. 5).

A novel approach for amplifying DNA that utilizes the Cas9 nickase-based amplification reaction (Cas9nAR) technique, allows for the simultaneous amplification of Salmonella typhimurium and Escherichia coli, two prevalent foodborne pathogens. This innovative method is capable of dual-target detection without the need for specialized equipment. The entire process can be completed in just three hours showcasing exceptional specificity and sensitivity. The detection limits for genomic DNAs and bacteria are both set at 100 copies and 100 CFU/mL, respectively. The results are visually displayed on a lateral flow strip [26,131]. CRISPR/Cas9 technology has found another use in detecting gene mutations. An assay with high sensitivity is designed to identify deletions in the epidermal growth factor receptor gene (EGFR) [132]. This approach aims to increase the presence of rare mutant alleles by removing wild-type fragments through the CRISPR complex. Subsequently, the mutant fragments are selectively amplified via PCR to achieve the detection sensitivity required for NGS. The results indicate that CRISPR/Cas9 in conjunction with PCR amplification can effectively enrich a variety of EGFR exon19 deletion mutations [132]. This may have implications for early cancer screening [133,134]. It is also possible to induce mutagenesis to mitigate resistance to cancer therapy [135,136]. Moreover, the combination of CRISPR/Cas9 and aptamers holds significant potential for applications in biotechnology and medicine. Aptamer mediated delivery of the CRISPR/Cas9 system is a delivery method that may reduce off-target effects. This method involves the modulation of CRISPR sgRNA through aptamers. For example, an investigation by Kundert et al. analyzed the possibility of regulating sgRNA using aptamers to produce ligand-responsive sgRNAs that activate and deactivate CRISPR/Cas9 function with theophylline [137]. Lin et al. also devised a comparable approach with small molecule-activated allosteric aptamer regulating (SMART) sgRNAs [138]. Aptamers in combination with CRISPR/Cas9 offer a promising solution to the lack of cell specificity for in vivo tumor delivery because of their impressive sensitivity and specificity, and minimal immunogenicity [139–141].

In addition to Cas9, dead Cas9 (dCas9) without nuclease activity is also used for nucleic acid detection. The innovative paired dCas9 (PC) detection platform was established with dCas9 [56]. Following amplification, the targeted DNA binds with dCas9 to form an active recombinant luciferase, which generates a fluorescent signal for detection. The PC detection system can be used to detect Mycobacterium tuberculosis DNA. Another rolling circle amplification-CRISPR-split-HRP (RCH) system based on dCas9 detection uses a pair of dCas9 to fuse the two domains of split-HRP (sHRP) after the target miRNAs are amplified by RCA technology [57]. Finally, the miRNAs are detected by adding the chromogenic substrate tetramethyl benzidine (TMB). The RCH system is mainly used to detect miRNAs. Detection methods accomplished through dCas9-mediated techniques cover a wide range of disciplines, including electrochemistry, fluid dynamics, and microfluidics. The CRISPR-Chip, a handheld device utilizing a graphene-based field-effect transistor (gFET) sensor combined with dCas9, enables quick and specific on-chip electrical tests of unamplified target genes within genomes without the need for amplification [58].

Previous restriction enzymes (REs) only recognized specific DNA sequences, while wild-type CRISPR/Cas9 nucleases require the PAM sequence. Consequently, current in vitro DNA cleavage and molecular cloning techniques remain incapable of precisely cleaving the directly neighboring target DNA base. A CRISPR-SpCas9 mutant, SpRY, which has no PAM sequence requirement, can cut DNA in almost any sequence on the genome [142]. In 2022, Walton et al. performed SpRYDNA digestion (SpRYgests) on multiple PAMs using over 130 gRNAs and found that SpRY can cut DNA of almost any sequence without PAM in vitro, including the hard-to-cut sites of wild-type SpCas9, which promises to simplify, accelerate, and improve the precision of molecular applications [142].

Cas12 is an essential RNA-guided endonuclease in gene editing second only to Cas9 in importance [143]. TracrRNA is crucial for the activity of Cas12b, while Cas12a functions independently of tracrRNA. However, more research is needed to fully understand the role of tracrRNA in Cas12c activity [38]. As illustrated in Fig. 6, Cas12 specifically recognizes target DNA under the guidance of gRNA, leading to specific cis-cleavage activity as well as non-specific trans-cleavage activity. This non-specific collateral cleavage activity is used in the development of nucleic acid detection technology.

The development of DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology and one-hour low-cost multipurpose highly efficient system (HOLMES) technology is rooted in the ssDNA trans-cleavage activity of Cas12. HOLMES was achieved through the utilization of the cis and trans-cutting functions of Cas12a, along with self-quenched-fluorescent ssDNA molecules as probes [59]. Chen et al. proposed the new detection method DETECTR by integrating the Cas12a trans-cleavage activity with IAT [60]. The process involves amplifying the target DNA through RPA or PCR, adding Cas12a and gRNA, and then introducing a fluorescence-quenched ssDNA probe. The Cas12a-gRNA complex targets and cleaves the DNA, triggering collateral cleavage activity which in turn cleaves the ssDNA probe, resulting in a fluorescent signal (Fig. 4A). DETECTR, based on CRISPR Cas12, offers a faster and more visually intuitive detection of SARS-CoV-2 using lateral flow chromatography [51]. DETECTR can also identify human papillomavirus (HPV) 16 and HPV18 within various subtypes of HPV [60].

The detection platform OR-DETECTR for identification of SARS-CoV-2 incorporates Reverse Transcription and Recombinase Polymerase Isothermal Amplification (RT-RPA) along with DETECTR technology [61]. In addition, a lateral flow assay was developed by Sun et al. based on OR-DETECTR for the detection of SARS-CoV-2. Operating through a lateral flow strip, the OR-DETECTR test offers a visual method of testing. The assay involves using activated Cas12a to cut the Carboxyfluorescein-biotin (FAM-biotin) reporter, generating a signal at the test line for initial detection. The uncut reporters are then captured at the control line. This design allowed for visual visualization of the trial results. The platform, which can be enclosed in a tube, is accurate, rapid, user-friendly, and does not require bulky equipment, making it suitable for at-home COVID-19 detection. During the COVID-19 outbreak in 2019, there was a critical need for accurate, rapid, and efficient testing methods. In response, scientists developed the all-in-one dual CRISPR/Cas12a (AIOD-CRISPR) method, which combines RPA technology with the Cas12a mechanism in a single-step testing platform [62]. Amplification and cutting are performed simultaneously in one system, which avoids contamination and reduces reaction time. AIOD-CRISPR produces visible fluorescence via the transverse cleavage ability of Cas12a, enabling the detection of SARS-CoV-2.

Another one-pot detection system is the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) platform, which is used in the SHERLOCK Testing in One Pot Covid, version 2 (STOPCovid.v2) assay. This one-pot assay platform integrates Cas12b with reverse transcription loop-mediated isothermal amplification (RT-LAMP) [63]. The procedure of STOPCovid.v2 is not complicated. First, viral nucleic acid is extracted, DNA extraction solution is added, and then RT-LAMP reagent and Cas12b reagent are added. When the reaction is over, the lateral flow test strip can be used for detection. The STOPCovid.v2 method does not need to extract viral RNA, nor does it need complex laboratory equipment. Operating this equipment is easy and quick, making it ideal for detecting SARS-CoV-2 in the field [72].

In order to make nucleic acid detection faster and more convenient, a suboptimal PAM of the Cas12a-based test (Cas12a-based sPAMC) was proposed in 2022. This method enables one-step CRISPR detection by IAT and Cas12a cleavage simultaneously in a single tube. By utilizing crRNAs that target substrates with suboptimal PAMs instead of standard PAMs, the reaction time is expedited by two to three times. The sPAMC approach can rapidly detect the human cytomegalovirus (HCMV) DNA virus within 10 min or the RNA virus SARS-CoV-2 in only 15 min using unextracted samples. Importantly, the sPAMC technique achieves detection limits comparable to traditional quantitative PCR (qPCR) in both cases [64].

The growing number of SARS-CoV-2 variants exhibiting spike mutations has sparked concerns due to increased transmission rates. Thus, a novel detection system for monitoring SARS-CoV-2 variants, known as Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS), was created. Upon binding of Cas12a/crRNA to the mutant target, the enzymatic cleavage activity of Cas12a is triggered, enabling non-specific cleavage of the single-stranded DNA reporter (FAM-ssDNA-BHQ or Biotin-ssDNA-Digoxin). Ultimately, the presence of the mutant target is identified through fluorescence detection or using a paper strip [144]. Cas12a in combination with the LAMP assay can also detect respiratory viral point mutations, including the mutant SARS-CoV-2 spike N501Y [145]. In 2022, a detection system called CRISPR Single Pot Assay for Detecting Emerging A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern (VOCs) (CRISPR-SPADE) was developed, which combines RT-LAMP and CRISPR in one reaction. The critical aspect of this approach lies in using a heat-resistant Cas12b effector endonuclease derived from Brevibacillus sp. BrCas12b demonstrates robust performance in an IAT buffer and thus integrating BrCas12b into an RT-LAMP based all-in-one reaction system is an effective method to differentiate SARS-CoV-2 VOCs in specimens from patients. This method enables continuous monitoring of the reaction in real time [65]. Compared with the detection of only one viral pathogen, the RPA/CRISPR-Cas12a system, when used in conjunction with lateral flow chromatography, enables the simultaneous identification of SARS-CoV-2, influenza A, and influenza B. This approach offers increased efficiency and effectiveness, serving as a robust tool for on-the-spot testing [66].

The use of CRISPR and similar technologies in cancer research has provided insight into previously challenging issues related to cancer genetics, the noncoding genome, and tumor diversity [146]. Ethical approval was granted in China for a 2016 clinical trial involving gene editing to address lung cancer, suggesting a potential breakthrough in developing an effective genome editing approach for combating cancer [147,148]. In 2021, the EasyCatch method for detecting mutations in cancer samples was developed. RPA technology was used to amplify the mutant sequences of cancer samples. The Cas12a-crNA complex first cleaved the target DNA after incorporating a fluorescence-quenched ssDNA probe and then cleaved the ssDNA probe to produce fluorescence [67].

Cas12a binds crRNA and the target DNA to form complexes and, upon Cas12a/crRNA binding to African swine fever virus (ASFV) DNA, this complex becomes activated and degrades the fluorescent ssDNA reporter in the system. When coupled with a point-of-care (POC) system, achieved a detection limit of 2 pM within 1 h without nucleic acid amplification [68]. Another detection technique that does not require amplification is Solid-Phase Extraction and Enhanced Detection Assay using CRISPR-Cas12a (SPEEDi-CRISPR). This method is based on using cas12a-coated magnetic beads to enrich target DNA, which can ultimately be read visually through smartphones or lateral flow analysis. SPEEDi-CRISPR can detect HPV18 and distinguish it from HPV16 and parvovirus B19. Additionally, it meets the requirements for point-of-care testing (Fig. 7) [69].

CRISPR/Cas12a’s trans-cleavage function was incorporated into an electrochemical biosensor (E-CRISPR), along with recombinase-aided amplification (RAA). By combining RAA and CRISPR, an electrochemical biosensor platform was developed for the identification of Listeria monocytogenes. This system shows a remarkable sensitivity with a detection threshold of 0.68 aM and exceptional specificity [70].

The CRISPR/Cas12c system requires the PAM sequence to bind the DNA in a target-specific fashion without cleavage. Using this feature, the Cas12c-based nucleic acid detection platform (Cas12c-DETECTOR) was developed. This platform was coupled to an optimized sgRNA for precise identification of single nucleotide polymorphisms (SNPs). By integrating pre-amplification techniques and either visual fluorescence detection methods or lateral flow strips, it can be employed for the detection of human and plant pathogens [71].

CRISPR proteins Cas12 and Cas13 have shown promising results in the detection and treatment of ssRNA viruses [65]. In particular, Cas13 can create a complex with numerous crRNAs, enabling targeted cleavage at various points along an RNA transcript with exceptional accuracy [149]. Cas13-crRNA complexes can be used to target specific sites within an ssRNA transcript, resulting in the cleavage of the RNA and subsequent inhibition of post-transcriptional gene expression. Cas13-mediated gene inhibition can counteract immune evasion mechanisms and enhance anti-cancer defenses [150].

Fig. 8 illustrates the activity of Cas13 in cleaving collateral RNA, with the Cas13-gRNA complex binding to the target RNA. This target RNA includes a specific protospacer-flanking site (PFS) located at the 3′ end [151]. By utilizing the collateral cleavage function of the CRISPR/Cas13 system, the nucleic acid detection method SHERLOCK was created. SHERLOCK offers quick and precise diagnostic tests relying on CRISPR/Cas13 for the detection of emerging COVID-19 [152]. SHERLOCK is similar to the DETECTR mechanism, but the target recognized by Cas13a is RNA, so when the target is DNA, the DNA needs to be transcribed into RNA for detection [73]. The initial step involves IAT of the target DNA using RPA technology, followed by transcription of the target DNA into target RNA by T7 transcript in vitro. When the Cas13a-crRNA complex is added, the non-specific trans-cleavage activity of Cas13a is triggered at the same time as its binding to the target RNA. The added fluorescent reporter RNA molecular probe is cleaved to produce a fluorescent signal (Fig. 4D). The SHERLOCK nucleic acid detection technology has proven to be effective in detecting ZIKV and dengue virus (DENV). Since the SHERLOCK platform can only detect a single nucleic acid at a time, the second-generation SHERLOCK (SHERLOCK v2) platform was created that can simultaneously detect four different target RNAs. In addition, a CRISPR-related enzyme (Csm6) was added to improve the sensitivity, and the test strip of the SHERLOCKv2 platform was further developed to detect the results intuitively [72].

To enhance the SHERLOCK platform’s detection capability, a technique called heating unextracted diagnostic samples to obliterate nucleases (HUDSON) was designed. This method allows for the direct release of viral nucleic acids from clinical samples for detection, rather than relying on conventional nucleic acid extraction methods [74]. Combining the SHERLOCK platform with HUDSON technology could directly detect DENV and ZIKV in blood, plasma, serum, saliva, and urine samples. Besides the above applications of Cas13, a SHERLOCK nucleic acid test was also constructed in 2019 to detect human Epstein-Barr virus (EBV) DNA [153]. The principle is that the activation of the Cas13 enzyme upon binding to the targeted viral RNA leads to the cleavage of the RNA and triggers collateral RNase activity [154]. This innovative platform provides a promising solution for efficient and accurate diagnosis of SARS-CoV-2, particularly in settings where immediate testing is required.

The SHERLOCK method is not only capable of detecting viruses but can also be used for malaria diagnosis [155,156]. By combining RPA technology, CRISPR-RNA base pairing, and Cas13a activity, SHERLOCK can detect all known Plasmodium species that cause malaria disease. Furthermore, SHERLOCK can be used to detect and drug-resistance genotyping in mosquitoes [156]. Lee et al. showcased the effectiveness of the SHERLOCK technique in detecting and differentiating different Plasmodium parasites [157]. They employed portable fluorescence detection or lateral flow strip detection methods, which are more intuitive and suitable for immediate detection.

SHERLOCK technology can also be used for the swift identification of soybean crop traits by amplifying and detecting nucleic acid targets from crude soybean extracts. The SHERLOCK technique is easier to operate than other detection methods, processes samples more quickly, and does not require complex instruments. These principles can also be extended to other agricultural detection methods [33].

The Cas13a protein has been enhanced for nucleic acid identification through the collateral lytic function of Leptotrichia wadei Cas13a (LwCas13a). A method called CRISPR/Cas13a-LFD was developed for quickly and accurately detecting ASFV by integrating RAA with a lateral flow strip [75]. CRISPR/Cas13a-LFD displayed precise detection abilities and can be used for on-site clinical ASFV identification with no expensive equipment. An alternative CRISPR/Cas13a trial exists that eliminates the need for amplification, enabling direct detection of SARS-CoV-2 in nasal swab RNA. This technology allows results to be interpreted using a smartphone microscope [76]. This method increases Cas13a activation by binding multiple crRNAs and analyzes fluorescence changes over time, offering a rapid, precise, compact, and economical solution for POC SARS-CoV-2 screening.

A platform developed by combining the collateral cleavage properties of Cas12a and Cas13a with HRP as an enzymatic reporter can generate signals detected by colorimetric, fluorescent, or chemiluminescent methods. This method, which does not require amplification, can detect both nucleic acid and non-nucleic acid targets and is a powerful dual-amplification sensing platform (Fig. 9). This technology has shown promise in quickly detecting SARS-CoV-2 viral nucleic acid markers [77].

Class II CRISPR/Cas systems demonstrate a wide range of structures and functions, offering flexibility in genome editing and nucleic acid detection [72]. The efficient Cas13d effectors display strong cleavage of targets and collateral RNase activity. The system, derived from Ruminococcus flavefaciens XPD3002, belongs to Class II type VI-D CRISPR/Cas13d [47,158]. The prophylactic antiviral CRISPR in human cells (PAC-MAN) technology has been reported to be useful against SARS-CoV-2, influenza A virus (IAV), and possibly all coronavirus strains [159]. There is a report of a CRISPR live-cell FISH tool based on Cas13d [160]. In this method, they used catalytically deactivated Cas13d and dCas13d defused with crRNA labeled with fluorescence [161].

In 2021, researchers created a technique to analyze SARS-CoV-2 and its altered forms using a method that uses RNA aptamers to signal CRISPR/Cas13 amplification [78]. Specificity of sequences was achieved through the ligation process and recognition by Cas13a/crRNA, enabling the detection of mutations in viral RNA. RNA aptamers that light up are unique RNA molecules capable of binding specifically to dyes and stabilizing their structure to create a complex with a fluorescent signal. The unlabeled RNA aptamer that lights up enables the sensitive generation of amplification signals through the ribonuclease activity of CRISPR/Cas13a triggered by the target [162,163].

The PCR-CRISPR platform is a Cas13a-based platform for Hepatitis B virus DNA (HBV) that can detect one copy of each HBV DNA and drug-resistant mutations [79], making it a valuable tool for early detection of HBV infection, monitoring drug resistance, and providing guidance for treatment.

Cas13a has shown potential in detecting miRNA biomarkers for early cancer diagnosis. A portable ECL chip (PECL)-CRISPR platform for detecting miRNAs was constructed by the introduction of Cas13a electrochemiluminescence (ECL) [80]. The target miRNAs were directly identified by Cas13a, and then the reaction was placed on an electrochemical luminescence to detect miRNAs in tumor cells on an electrochemical luminescence chip [164]. This technology offers a promising approach in the field of clinical diagnostics.

The Cas14 protein requires tracrRNA and crRNA to identify and target ssDNA without the need for a PAM site and activates Cas14 protein-specific cis-cleavage activity as well as non-specific collateral cleavage activity (Fig. 10). While Cas14 primarily targets ssDNA, it can also target dsDNA through a T-rich PAM sequence [165,166]. Based on the characteristics of Cas14, Cas14a-DETECTR technology was developed for the identification of human high-fidelity DNA SNP sites and viral DNA (Fig. 4B) [48]. Integrating the CRISPR/Cas14a system with 2D porphyrin metal-organic framework nanosheets (2D-pMOFs) has led to the creation of a novel and adaptable fluorescence sensor known as the Cas14-pMOFs fluorescence sensor, which allows for highly selective and sensitive detection of microcystin-LR (MC-LR) levels [81].

By integrating Cas14a1 with asymmetric PCR, Hu et al. created a technique to identify homozygous deletion of exon 7 of the survival motor neuron 1 gene (SMN1) in patients with spinal muscular atrophy (SMA). In this testing method, specific crRNA-guided Cas14a1 endonuclease amplifies target DNA. Once activated, Cas14a1 breaks down the ssDNA-FQ probe via non-specific trans-cleavage activity, leading to the production of a fluorescence signal [167]. The engineered effector CasMINI, based on the naturally found Cas12f (Cas14) system, was created using CRISPR/Cas technology. Experimental findings revealed that while the original Cas12f and its corresponding sgRNA were inactive in mammalian cells, the modified Cas12f protein and sgRNAs demonstrated effective control over gene expression and editing, offering the potential for genome engineering tasks [42]. Compared to Cas9, Cas14 has the advantage of targeting a wider range, but whether this might cause higher off-target effects is yet to be addressed.