Using Split CRISPR/Cas12a Technology to Improve Molecular Testing and Biosensors

Split technology in CRISPR/Cas12a-based systems revolves around artificially dividing critical nucleic acid elements—such as crRNAs or activator strands—into two or more fragments that, when combined, are inactive but reconstitute function upon target-induced assembly or in response to specific triggers [9,10]. Canonically, Cas12a operates with a single crRNA composed of a 5′-handle (scaffold) and a 3′-spacer region that together guide the enzyme to a complementary nucleic acid target, triggering target-specific (cis) cleavage as well as robust collateral (trans) cleavage of single-stranded DNA reporters [9,11]. In split designs, this highly orchestrated interaction is conditionally controlled, allowing precise regulation over when and how the sensor is activated.

A primary approach involves separating the crRNA into distinct moieties: the scaffold (or repeat) and the spacer [11]. The scaffold is invariant and responsible for Cas12a binding and structural stability, while the spacer provides programmable target specificity [11]. When these elements are supplied separately, only their successful co-localization and hybridization—often mediated or facilitated by the target nucleic acid itself—restore the canonical crRNA structure, thus reconstituting the Cas12a activation potential [12]. In addition, the crRNA can be further engineered: "splice-at-will" strategies allow for the crRNA to be split at nearly any point within the spacer, generating truncated crRNA (tcrRNA) that can remain inactive unless an auxiliary (often target-derived) RNA or a synthetic activator bridges the gap, completing the functional guide RNA [10]. Beyond crRNA, split activator designs have emerged as powerful alternatives, especially for target-induced assembly [9]. In this format, the traditional single-stranded DNA (ssDNA) or RNA activator sequence, essential for initiating collateral cleavage, can be divided into two or more fragments [9]. These fragments may reassemble in response to a specific nucleic acid analyte, chemical reaction, or hybridization event, generating the active form that triggers Cas12a's trans-cleavage activity [13]. These modular splits greatly enhance detection flexibility and enable conditional, "AND-gate" style activation pathways for integrated biosensing and logic functions [9].

Split systems utilize proximity effects and molecular recognition to convert the presence of an analyte into sensor activation [13]. For split crRNA systems, one critical mechanism is target-assisted assembly, whereby the presence of a complementary DNA or RNA causes the two crRNA fragments (scaffold and spacer) to associate, correctly orienting them for Cas12a binding and activation [11]. The efficiency and specificity of this reassembly depend on carefully optimized hybridization stability, fragment lengths, and sequence complementarity [10]. In "splice-at-will" designs, truncated crRNAs with a missing spacer region are complemented by exogenous RNAs, such as ultrashort miRNAs or application-specific targets, forming a complete crRNA sequence that prompts robust Cas12a activation [10]. For split activator systems, either a chemical reaction (e.g., hydrazone bond formation), DNA strand displacement, enzymatic cleavage, or a simple target hybridization event can unite the split parts into a full-length activator [13]. Hydrazone chemistry offers a striking example: two activator fragments, modified with hydrazine and aldehyde groups, respectively, remain inert until brought into proximity—often by a target-induced strand displacement—at which point they form a hydrazone bond, reconstructing the canonical activator and thus initiating the Cas12a response [13]. This chemical proximity effect simultaneously accelerates assembly and enriches specificity, as even single-nucleotide mismatches within the target can strongly inhibit activator formation and subsequent signaling [13].

A key advantage of split technology is its capacity for highly tunable sensor responses [9]. The assembly parameters—such as redundancy (extra nucleotides at the split junction), split site, fragment concentration, and hybridization domains—can be meticulously programmed to achieve fine-grained control over Cas12a activity [9]. For example, introducing mismatches, short gaps, or overhangs at the junction between tcrRNA and its complementary RNA can dramatically modulate, or even abrogate, Cas12a trans-cleavage, affording exquisite sequence discrimination [10]. Split activator systems can leverage redundancy and position-dependent structures to provide an adjustable activation threshold, supporting complex logic operations and facilitating the integration of upstream molecular networks, such as DNAzymes or aptamer assemblies [9]. The specificity imparted by split designs is frequently superior to that of conventional non-split systems, as only the correct assembly of all split components—often relying on perfect sequence complementarity—restores functional activation; thus, off-target or background cleavage is inherently minimized [11,13]. Likewise, by requiring target-induced reassembly, split technology can distinguish between highly similar sequences, such as mature and precursor miRNAs, or discriminate single-nucleotide variants crucial for precise diagnostics [10].

The principles established by split technology enable much more than conventional biosensing [14]. Beyond straightforward detection, split CRISPR/Cas12a components function as molecular logic gates ("AND," "OR," "NOT," "NAND"), where sensor activation occurs only when precise molecular-input conditions are satisfied, such as the simultaneous presence of two different nucleic acids or enzymes [9]. Modular platforms utilizing these mechanisms have demonstrated simultaneous detection of different SNPs, enzyme activities, and integration within multilayered molecular reaction networks [9]. This approach supports seamless multiplexing, where each unique pair of split fragments may be selectively activated by a specific target, reducing cross-talk and enhancing assay scalability [11].

Detailed structural and biochemical studies reveal that the split RNAs or activators, upon correct assembly, closely mimic the native conformation required for high-affinity Cas12a binding and activation [10]. For split crRNAs, the hybridized scaffold-spacer mimics the full-length crRNA: experimental evidence shows near-equivalent rates of cis- and trans-cleavage compared to the wild-type system, provided the split is optimally designed, and hybridization is efficient [10,11]. Preassembly conditions, fragment ratios, and sequence optimization are crucial for achieving native-like activity and strong signal outputs [10].

In summary, the underlying principles of split technology in CRISPR/Cas12a are grounded in molecular engineering that enforces strict analyte dependence for functional assembly, capitalizing on proximity effects, hybridization fidelity, and controlled chemical reactions to translate target presence into robust and specific sensor responses [11,13]. These strategies offer versatile, modular, and highly programmable platforms for biosensing, diagnostics, and bio-computational logic, positioning split CRISPR/Cas12a systems among the most adaptable emerging technologies in synthetic biology and molecular diagnostics [9,10].

The engineering of split systems in CRISPR/Cas12a-based sensors is fundamentally centered on increasing the programmability, specificity, and functional versatility of CRISPR diagnostics by dividing critical nucleic acid components into multiple fragments that require conditional assembly for activity [10]. The most widely employed strategy involves splitting the crRNA, which guides Cas12a to its target, or the activator sequence, into discrete segments that alone are inactive but regain functionality when brought together by target-specific interactions or external triggers [11]. This modular assembly leverages advances in nucleic acid nanotechnology and chemical biology to finely regulate Cas12a activation in response to diverse molecular cues [9,14].

A prominent and foundational approach is the bifurcation of the Cas12a crRNA into its structural domains: the handle-like repeat region (scaffold) and the variable target-recognition region (spacer) [11]. In this split crRNA strategy, the 20-nucleotide (nt) scaffold RNA and the 20-nt spacer RNA are provided separately [15], allowing for flexible reconstitution of the active crRNA only when both segments are present and correctly hybridized [11]. This design is highly programmable, as different spacers can be matched with a universal scaffold to target multiple sequences, enabling straightforward multiplexing and rapid adaptation to new targets [11]. Structural studies confirm that, upon hybridization, the scaffold and spacer functionally mimic the native crRNA, restoring both target binding and trans-cleavage activities of Cas12a with comparable efficiency to wild-type systems when optimally designed [11].

Innovative "splice-at-will" engineering further extends the split concept by allowing the crRNA to be cleaved at almost any site within the spacer region [10]. This results in a truncated crRNA (tcrRNA) that, by itself, is functionally inert but which regains activity upon hybridization with ultrashort target RNAs (as short as 6–8 nucleotides), thereby forming a functional crRNA capable of activating Cas12a [10]. This strategy is especially valuable for the direct detection of ultrashort microRNAs (miRNAs), enabling acceptable discrimination of single-nucleotide differences through precise control of the reassembly site and RNA complementarity [10]. The flexible truncation site design also allows tailoring the system for sequence specificity, sensitivity, and adaptability to many types of RNA targets [10].

Split activator engineering involves dividing the canonical activator—typically a single-stranded DNA (ssDNA) or hybrid DNA-RNA strand—into two or more separate fragments [16]. Each fragment alone is incapable of activating Cas12a, but reconstitution through target-induced hybridization, chemical ligation, or logic-gate-like assembly restores the full activator, thereby triggering robust trans-cleavage activity [16]. For example, the split nucleic acid-activated Cas12a (SNA-Cas12a) system exploits this principle for direct miRNA detection, where a short single-stranded DNA (ssDNA) is added to the crRNA spacer to facilitate direct RNA recognition and enhancer strand binding [16]. Chimeric DNA-RNA hybrid activators further expand versatility by utilizing fragments of both nucleic acids, which can be conditionally assembled by the target [16]. Hydrazone chemistry-mediated split systems exemplify a powerful chemically inducible strategy [13]. Here, two split activator strands are designed to have reactive chemical groups (such as hydrazine and aldehyde) at their termini. Only upon target-induced proximity, often through hybridization or a strand displacement reaction, are these fragments chemically ligated into a full-length activator, thus providing both spatial and chemical specificity while simultaneously enhancing selectivity for single-nucleotide mismatches [13].

Recent variants introduce spatial constraint-based control by engineering split crRNA systems that can be transiently blocked through the introduction of macromolecules or chemical modifications at the 3′ end of the scaffold [14]. In spatially blocked split CRISPR-Cas12a (SBS-Cas) systems, the 3′ end is chemically modified, such as by conjugating small molecules like biotin or acrylamide [14]. Macromolecules (e.g., streptavidin, antibodies, DNA strands) bind to these modifications, exerting spatial hindrance that occludes proper assembly of the scaffold with Cas12a and renders the system inactive [14]. Upon introduction of a free small molecule analyte (such as biotin, m6A, or glutathione), competitive binding displaces the blocking agent, releasing the scaffold to assemble with Cas12a and thus activating the biosensor [14]. This modular design offers low background signals, enhanced adaptability to various small molecule targets, and ultra-sensitive quantitative detection [14,17].

Advancements in split engineering are increasingly integrated with various detection modalities (fluorescence, lateral flow, electrochemical) and coupled with amplification techniques (e.g., RPA, isothermal, or zero-amplification formats) to produce robust, field-deployable diagnostics (Figure 1) [11]. The modularity and orthogonality of split designs further enable scalable multiplexing and parallel detection of multiple targets with reduced cross-talk [18]. The evolution of split system engineering in CRISPR/Cas12a-based sensors has introduced a spectrum of variants—ranging from canonical scaffold-spacer splits to chemically mediated and spatially blocked systems—that collectively offer unprecedented programmability, specificity, and adaptability for both nucleic acid and small molecule biosensing [11,14]. By leveraging these strategies, researchers can design next-generation molecular diagnostics with broad applications in clinical, environmental, food safety, and bioimaging fields [2,14].

The CRISPR-Cas12a system, a powerful RNA-guided endonuclease derived initially from bacterial immune systems, is utilized widely for nucleic acid detection and genome editing. Its unique ability to specifically target and cleave DNA, along with a mechanism that indiscriminately degrades non-targeted single-stranded DNA (ssDNA), makes it particularly effective for rapid diagnostics [19,20]. There are two primary configurations of the Cas12a system: the complete Cas12a system and the engineered split Cas12a system, each offering distinct advantages and disadvantages in terms of performance metrics such as sensitivity, specificity, detection time, and scalability [21,22,23]. The complete Cas12a system operates as a full-length enzyme guided by CRISPR RNA, forming a 20-nucleotide R-loop to recognize and cleave target DNA. Its trans-cleavage activity enhances sensitivity by facilitating indiscriminate cleavage of non-specific ssDNA, aiding in signal amplification. This system is notable for its rapid kinetics and efficiency, making it suitable for genome editing and swift nucleic acid detection [21,22,23]. Conversely, the split Cas12a system is designed with the Cas12a protein divided into inactive fragments that only become active when specific conditions, such as the presence of target nucleic acid, induce their reassembly. This mechanism significantly reduces background activity and enhances specificity, minimizing false positives. However, the added kinetic steps involved in reassembly can lead to slight trade-offs in detection speed. Still, when paired with amplification techniques, split Cas12a systems exhibit high sensitivity, making them ideal for applications requiring precise discrimination, such as identifying single-nucleotide polymorphisms (SNPs). In terms of sensitivity and detection capabilities, complete Cas12a systems excel, detecting targets in the picomolar range and achieving even greater sensitivity when coupled with amplification techniques like RT-LAMP. They have demonstrated the ability to identify low viral loads in clinical settings [21,22,23]. Similarly, split Cas12a systems also achieve high sensitivity, particularly when combined with amplification strategies, benefiting from their controlled activation for precise applications. Ultimately, the choice between a complete or split Cas12a system hinges on the specific requirements of the application, balancing performance characteristics to optimize outcomes in diagnostics and genome editing [19,20,21,22,23]. The following table provides a detailed comparison of these two systems [22] (Table 1).

Adequate sample preparation is paramount for the successful deployment of Cas12a-based assays in clinical settings [33]. For saliva samples, which are non-invasive and easily collected, protocols often focus on minimal preprocessing to maintain user-friendliness and speed [33]. Chemical treatments coupled with heat inactivation effectively stabilize viral RNA, enabling direct detection without the need for RNA extraction, which is typically a bottleneck in traditional molecular diagnostics [29,33]. This extraction-free approach not only simplifies the workflow but also maintains high sensitivity and specificity, with reported agreements exceeding 97% compared to traditional RNA extraction methods for SARS-CoV-2 detection [29,30]. The detection of high viral loads in saliva has also been observed, further validating its utility as a diagnostic specimen [29,30]. Blood samples present greater complexity due to the presence of various cellular components, proteins, and inhibitors like hemoglobin, which can interfere with enzymatic reactions. Therefore, optimized chemical treatments and controlled heat inactivation are crucial to balance RNA preservation and viral inactivation [30,34]. These pre-treatment steps enable direct assay application while mitigating false positives arising from matrix effects. Integrated microfluidic systems are particularly beneficial here, streamlining the preparation steps and leading to efficient assay performance [28,34].

Clinical samples like blood and saliva contain a variety of biomolecules, enzymes, and endogenous inhibitors that can significantly impair the performance of nucleic acid detection assays [35]. These inhibitors can degrade target nucleic acids or impede the activity of enzymes crucial for Cas12a's collateral cleavage function, potentially reducing sensitivity and specificity [35]. The intrinsic design of split Cas12a offers a robust solution to these issues [32]. By requiring the reassembly of inactive fragments for activation, the system inherently reduces background cleavage and minimizes false positives, thereby improving the signal-to-noise ratio in complex biological matrices [32]. This conditional activity means that the collateral function is less likely to be triggered by non-specific interactions or endogenous components within the sample [24,32]. Furthermore, strategies such as optimized buffer compositions and the inclusion of specific cofactors, like manganese ions, significantly enhance the sensitivity and accelerate the collateral cleavage kinetics of Cas12a, compensating for potential delays due to fragment reassembly and maintaining rapid detection times even in the presence of inhibitors [24]. This synergistic approach allows for the sensitive and specific detection of low-copy nucleic acids directly from minimally processed saliva and blood [24].

The development of point-of-care (POC) diagnostics is a primary focus in clinical applications to enable rapid, decentralized testing [31,36]. Split Cas12a assays are highly amenable to integration into POC devices due to their potential for simplified workflows and robust performance in minimally processed samples. Key protocols for integrating split Cas12a assays into POC devices for blood and saliva include Minimal Fluid Handling: Designs favor devices that require minimal manual steps, reducing the chance of human error and cross-contamination [31,36]. Single-tube or cartridge-based systems are ideal. Isothermal Reaction Conditions: Many Cas12a applications are compatible with isothermal amplification methods (e.g., RT-LAMP, RAA), which eliminate the need for complex thermocycling equipment, simplifying device design and power requirements [32,33]. Portable Readout Systems: Integration with smartphone-based fluorescence readouts or lateral-flow assays makes detection accessible and user-friendly, supporting deployment in resource-limited settings [29,37]. For instance, the CASSPIT system uses CRISPR-Cas13a-based SARS-CoV-2 detection with a lateral-flow assay readout, showing 98% agreement with RT-qPCR data for clinical samples, and integrates with a smartphone application for user-friendly results [29]. Reagent Stability: Ensuring the long-term stability of split Cas12a components, particularly their ability to reassemble efficiently, is critical for POC applications. Optimized buffer systems and lyophilized reagents contribute to extended shelf life [32,33]. These integrations aim to make highly accurate molecular diagnostics available outside traditional laboratory settings, which is crucial for disease management and outbreak control [31].

The demand for widespread and rapid testing, particularly during public health crises like the COVID-19 pandemic, highlights the need for scalable and high-throughput diagnostic solutions [33,38]. Split Cas12a assays, like their complete counterparts, are increasingly being integrated into automated platforms to address this need [28]. Integrated microfluidic systems, such as centrifugal microfluidic platforms, can automate the entire diagnostic process from sample preparation to detection [28]. The CASMEAN system, for example, integrates recombinase-aided amplification (RAA) with the Cas12a system to rapidly detect nucleic acids within 1.5 h, making it suitable for high-volume testing in clinical laboratories [28,39]. Automation significantly reduces hands-on time and operational complexity, allowing for the processing of numerous samples concurrently [39]. This is particularly important for high-volume blood and saliva testing, where the rapid turnaround of results can significantly impact public health efforts [38]. The non-invasive nature of saliva collection makes it an attractive specimen for large-scale screening and population-based surveillance [38,39]. Pooled sample testing, where multiple saliva samples are combined and tested together, can further increase testing capacity, especially in low-prevalence settings [38]. However, it requires careful consideration of sensitivity reduction for weakly positive samples. Split Cas12a's high specificity makes it well-suited for such pooling strategies by minimizing false positives that could arise from non-specific reactions in pooled complex matrices.

The enhanced specificity of split Cas12a technology, stemming from its conditional activation mechanism, makes it particularly advantageous for multiplexed detection in clinical samples [24]. Multiplexing allows for the simultaneous detection of multiple pathogens or biomarkers within a single sample, providing a comprehensive diagnostic profile and conserving resources [24]. The ability of split Cas12a, especially variants like MeCas12a, to distinguish between single nucleotide polymorphisms (SNPs) with high fidelity is critical for applications such as:

Pathogen Variant Identification: Differentiating between closely related viral strains or bacterial species is crucial for epidemiological tracking and guiding treatment strategies [24].

Genotyping: Identifying genetic mutations or variations associated with disease susceptibility, drug resistance, or personalized medicine [24].

Biomarker Panels: Simultaneously detecting multiple disease biomarkers from a single blood or saliva sample to improve diagnostic accuracy and provide a more complete clinical picture [31]. The modular nature of split Cas12a components lends itself to complex assay design, enabling the development of sophisticated multiplexed formats that require high specificity and minimal cross-reactivity between different targets [32]. This capability significantly enhances the utility of Cas12a in diverse clinical diagnostic scenarios, from infectious diseases to genetic screening [32].

The advent of CRISPR/Cas systems has revolutionized molecular diagnostics, offering powerful tools with remarkable specificity and versatility [40]. Among these, split CRISPR/Cas12a technology has emerged as a particularly promising avenue, enhancing the practicality of diagnostic assays in diverse real-world contexts, particularly within clinical settings [41,42]. The modular design and conditional activation mechanisms of split systems enable highly sensitive, specific, and rapid detection of a wide array of targets, from nucleic acids to proteins, making them ideal for point-of-care (POC) applications. The performance of split CRISPR/Cas12a systems is defined by key metrics: sensitivity, specificity, and detection speed [15,42]. These systems demonstrate impressive sensitivity, with limits of detection (LoD) often reaching attomolar or femtomolar concentrations, even without pre-amplification steps. For instance, a Framework-Hotspot reporter (FHR) enhanced CRISPR-Cas12a platform achieved sensitive detection down to 100 fM for pathogen nucleic acids and tumor protein markers, with results available in under 15 min [15,42,43]. Another split Cas12a system (SCas12a) demonstrated an LoD of 10 fM for miRNA detection, again without the need for reverse transcription or pre-amplification. When combined with amplification techniques like recombinase polymerase amplification (RPA), sensitivity can be further boosted, allowing detection of targets like human papillomavirus (HPV) at attomolar concentrations [15]. Specificity is another hallmark of split CRISPR/Cas12a systems. The precise coordination required for the reassembly of split components ensures minimal off-target activity, critical for accurate diagnostics [15]. These systems can effectively discriminate between highly similar sequences, including distinguishing mature miRNAs from their precursors and identifying single-nucleotide polymorphisms (SNPs) [44]. For example, the SCas12aV2 assay, an enhanced split Cas12a system, exhibits high specificity for SNPs and successfully identifies SARS-CoV-2 infections [44]. Similarly, a recent RPA-CRISPR/Cas12a-LFS method designed for Duck circovirus (DuCV) detection showed 100% consistency with real-time quantitative polymerase chain reaction (qPCR) and no cross-reactivity with six other avian viruses, confirming its high specificity [45]. Speed is paramount in clinical diagnostics, and split CRISPR/Cas12a systems offer rapid turnaround times. Many platforms deliver results within minutes to an hour. The RPA-CRISPR/Cas12a-LFS system for DuCV detection yields results in just 45 min [45]. Another method for miRNA detection, based on a split-T7 switch, can achieve femtomolar detection within 1 h. Moreover, Cas12a-Graphene Field-Effect Transistor (GFET) devices can differentiate positive cases from serum samples within 5 min [46]. The robust performance of split CRISPR/Cas12a technology translates directly into practical applications for clinical sample analysis. These systems have been successfully validated for detecting various biomarkers and pathogens across a spectrum of human and animal samples, including blood, serum, saliva, urine, nasopharyngeal swabs, and tracheal aspirates [47,48,49]. The ability to perform sensitive and specific detection directly from complex matrices, often without extensive pre-processing or amplification, significantly enhances their utility in point-of-care settings, reducing the need for expensive equipment and specialized personnel [46].

This Table 2 illustrates the diverse applications and robust performance of split CRISPR/Cas12a systems in clinical sample diagnostics. They offer high sensitivity crucial for early disease detection, exceptional specificity to prevent false positives, and rapid detection times, which are vital for timely intervention [46]. The flexibility in sample types, ranging from easily accessible saliva and urine to more complex blood and tissue aspirates, further underscores their practical applicability in various diagnostic settings. Such attributes make split CRISPR/Cas12a a formidable tool, addressing the pressing need for rapid, sensitive, and user-friendly diagnostic platforms in modern healthcare. The development of split CRISPR/Cas12a technology represents a significant stride in molecular diagnostics. Its inherent advantages—high sensitivity, robust specificity, and swift detection capabilities—make it profoundly practical for widespread clinical adoption. The successful application across a multitude of clinical sample types, often without complex pre-amplification steps, positions these systems as critical components for next-generation point-of-care testing and accessible precision medicine. As research continues to refine these systems, their role in disease surveillance, early diagnosis, and personalized treatment strategies is poised to expand even further.

Current iterations of split CRISPR/Cas12a systems exhibit diminished efficiency and sensitivity when addressing RNA with intricate secondary structures, which poses challenges in clinical contexts. The goal is to develop and validate universal scaffold and activator designs tailored for various structured RNA targets, while integrating machine learning approaches to predict optimal split crRNA configurations. Despite advancements like SCas12aV2, structured RNA detection remains a bottleneck, necessitating solutions to overcome steric hindrance for broader applicability in clinical diagnostics 9/9/202552.

The multiplexed detection capabilities of pooled split crRNAs face notable challenges concerning signal interference and cross-reactivity, which impede the scalability of assays. Solutions include the design of orthogonal split crRNA sets that exhibit minimal cross-talk, paired with the development of computational tools for the efficient design of multiplex assays, followed by experimental validation in complex biological samples. While multiplexing is pivotal for cost-effective diagnostics, precise design is essential to minimize false positives and maintain assay sensitivity [52,53].

Split CRISPR/Cas12a assays devoid of amplification often reveal reduced sensitivity when applied to clinical or environmental samples due to matrix effects. Approaches to address this issue involve engineering split crRNA and Cas12a variants that exhibit enhanced stability and activity in complex matrices, alongside the development of sample preparation methods that align with amplification-free detection protocols. Given the necessity for high sensitivity without amplification in real-world scenarios, this area remains underexplored [54,55].

Variability in the efficiency and stability of split crRNA-Cas12a complex reassembly across varied conditions undermines assay reproducibility. A systematic characterization of split crRNA assembly kinetics and stability under different environmental factors—such as temperature, ionic strength, and biological fluids—is essential. Establishing standardized protocols and reagents will form the foundation for reliable reassembly, a critical aspect that currently lacks comprehensive characterization [56].

Existing methods for activity control (e.g., RNA G-quadruplex, elongation-caged activators) introduce complexity and require optimization, thereby limiting practical deployment. The development of simplified, modular activity regulation strategies, compatible with split crRNA frameworks, is critical. Exploring light- or small molecule-induced switches for on-demand activation could enhance the specificity of Cas12a activity while simplifying assay design and operation [53,57,58].

While aptamer-based CRISPR/Cas12a assays demonstrate the capability to detect proteins and small molecules, their integration with split crRNA technology remains limited. Efforts should focus on engineering split crRNA-Cas12a platforms that incorporate aptamer or allosteric ribozyme modules, allowing for multiplexed detection of non-nucleic acid targets, with validation in clinical samples. Expanding the detection repertoire to non-nucleic acid targets enhances the diagnostic scope, but compatibility issues with split systems still need to be resolved [59,60,61].

Few studies have successfully leveraged split CRISPR/Cas12a systems for real-time or intracellular detection with low background noise. The advancement of split crRNA designs and delivery methodologies specifically for live-cell imaging and real-time monitoring is vital, alongside optimizing activation strategies that minimize background interference. Enhancing intracellular and dynamic detection will expand the applications of these systems but necessitates refined signal control and delivery methods [62,63].

The absence of generalized design rules for split crRNA sequences and activators hinders the efficient development of assays for novel targets. To address this, the creation of computational platforms that integrate both structural and biochemical data for predicting effective split crRNA designs is imperative; considerations for target secondary structure should also be incorporated. Streamlining the design process can facilitate faster assay development and reduce the trial-and-error phase [52,54].

The stability of split crRNA and Cas12a complexes during storage and transport has not been thoroughly investigated, which hampers their deployment in the field. Research focused on lyophilization and stabilization formulations for these components is necessary, along with assessments of activity retention over time under varying conditions. Ensuring stability is crucial for point-of-care applications and resource-limited settings, yet available data in this area remain sparse [64].

Most split CRISPR/Cas12a assays lack extensive validation across diverse clinical cohorts and sample types. Conducting large-scale clinical studies that compare the performance of split CRISPR/Cas12a assays against gold-standard diagnostic methods across various diseases and sample matrices will be essential. Comprehensive clinical validation is vital for transitioning promising assays into practical diagnostic tools [53,54].