Ultrasensitive detection of Salmonella typhi using a PAM-free Cas14a-based biosensor.

The effectiveness of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas14a1, widely utilized for pathogenic microorganism detection, has been limited by the requirement of a protospacer adjacent motif (PAM) on the target DNA strands. To overcome this limitation, this study developed a Single Primer isothermal amplification integrated-Cas14a1 biosensor (SPCas) for detecting Salmonella typhi that does not rely on a PAM sequence. The SPCas biosensor utilizes a novel primer design featuring an RNA-DNA primer and a 3'-biotin-modified primer capable of binding to the same single-stranded DNA (ssDNA) in the presence of the target gene. The RNA-DNA primer undergoes amplification and is blocked at the biotin-modified end. Subsequently, strand replacement is initiated to generate ssDNA assisted by RNase H and Bst enzymes, which activate the trans-cleavage activity of Cas14a1 even in the absence of a PAM sequence. Leveraging both cyclic chain replacement reaction amplification and Cas14a1 trans-cleavage activity, the SPCas biosensor exhibits a remarkable diagnostic sensitivity of 5 CFU/mL. Additionally, in the assessment of 20 milk samples, the SPCas platform demonstrated 100% diagnostic accuracy, which is consistent with the gold standard qPCR. This platform introduces a novel approach for developing innovative CRISPR-Cas-dependent biosensors without a PAM sequence.

CRISPR/Cas14 and G-Quadruplex DNAzyme-Driven Biosensor for Paper-Based Colorimetric Detection of African Swine Fever Virus.

The highly contagious nature and 100% fatality rate contribute to the ongoing and expanding impact of the African swine fever virus (ASFV), causing significant economic losses worldwide. Herein, we developed a cascaded colorimetric detection using the combination of a CRISPR/Cas14a system, G-quadruplex DNAzyme, and microfluidic paper-based analytical device. This CRISPR/Cas14a-G4 biosensor could detect ASFV as low as 5 copies/µL and differentiate the wild-type and mutated ASFV DNA with 2-nt difference. Moreover, this approach was employed to detect ASFV in porcine plasma. A broad linear detection range was observed, and the limit of detection in spiked porcine plasma was calculated to be as low as 42-85 copies/µL. Our results indicate that the developed paper platform exhibits the advantages of high sensitivity, excellent specificity, and low cost, making it promising for clinical applications in the field of DNA disease detection and suitable for popularization in low-resourced areas.

Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas enzymes have been widely applied for biosensor development, combined with various isothermal amplification strategies (IAS) to boost sensitivity and specificity. Currently, the unstable assay and tedious manipulation usually hinder its practical applications. Here, a Cas14a1-advanced LAMP assay (CALA) combined with Rapid Extraction of Bacterial Genomic DNA (REBGD) is proposed for pathogen detection. For rapid CALA, a single stranded fluorescence reporter and ssDNA-gold nanoparticles (AuNPs) are used as signal indicators to establish ultrasensitive and visual platforms. This assay displays precise detection of bacteria, which can achieve an ultrasensitive limit of detection (LOD) 10 aM target genomic DNA. Furthermore, the high reliability of pathogen diagnostic for contrived samples is validated through the rapid visual CALA platform, demonstrating the promising practical testing availability of pathogen detection.

Toehold-Containing Three-Way Junction-Initiated Multiple Exponential Amplification and CRISPR/Cas14a Assistant Magnetic Separation Enhanced Visual Detection of Mycobacterium Tuberculosis.

Rapid and simple nucleic acid detection is significant for disease diagnosis and pathogen screening, especially under specific conditions. However, achieving highly sensitive and specific nucleic acid detection to meet the time and equipment demand remains technologically challenging. In this study, we proposed a magnetic separation enhanced colorimetry biosensor based on a toehold-containing three-way junction (TWJ) induced multiple isothermal exponential amplification and the CRISPR/Cas14a (C-TEC) biosensor. The TWJ template was designed as a Y-X-Y structure. In the presence of the target, the formation of toehold-containing TWJ complex induced primer extension, leading to the generation of amplified single-stranded DNA; this amplified DNA could then bind to either the free TWJ template for EXPAR reaction or the toehold of the TWJ complex for toehold-mediated strand displacement, thereby enabling the recycling of the target. The amplification products could trigger CRISPR/Cas14a for efficient trans-cleavage and release the magnetically bound gold nanoparticle probes for colorimetry detection. Using Mycobacterium tuberculosis 16S rDNA as the target, the proposed C-TEC could detect 16S rDNA down to 50 fM by the naked eye and 20.71 fM by UV-vis detector at 520 nm within 90 min under optimal conditions. We successfully applied this biosensor to clinical isolates of Mycobacterium tuberculosis. In addition, the C-TEC biosensor also showed feasibility for the detection of RNA viruses. In conclusion, the proposed C-TEC is a convenient, fast, and versatile platform for visual detection of pathogen DNA/RNA and has potential clinical applications.

High-Fidelity Identification of Single Nucleotide Polymorphism by Type V CRISPR Systems.

Accurate and sensitive detection of single nucleotide polymorphism (SNP) holds significant clinical implications, especially in the field of cancer diagnosis. Leveraging its high accuracy and programmability, the CRISPR system emerges as a promising platform for advancing the identification of SNPs. In this study, we compared two type V CRISPR/Cas systems (Cas12a and Cas14a) for the identification of cancer-related SNP. Their identification performances were evaluated by characterizing their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a system exhibited superior accuracy and robustness over the CRISPR/Cas12a system for SNP detection. Furthermore, blocker displacement amplification (BDA) was combined with the CRISPR/Cas14a system to eliminate the interference of the wild type (WT) and increase the detection accuracy. In this strategy, we were able to detect BRAF V600E as low as 103 copies with a sensitivity of 0.1% variant allele frequency. Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to identify the BRAF mutation from human colorectal carcinoma cells, achieving a high sensitivity of 0.5% variant allele frequency, which is comparable to or even superior to those of most commercially available products. This work has broadened the scope of the CRISPR system and provided a promising method for precision medicine.

Multiple Isothermal Amplification Coupled with CRISPR-Cas14a for the Naked-eye and Colorimetric Detection of Aflatoxin B1.

Aflatoxin B1 (AFB1) is highly toxic and challenging to remove, posing significant risks to both human health and economic development. Therefore, there is an urgent need to develop rapid, simple, and sensitive detection technologies. In this study, we introduce a naked-eye and colorimetric method based on multiple isothermal amplifications coupled with CRISPR-Cas14a and investigate its biosensing properties. This technique utilizes composite nanoprobes (MAPs) comprising magnetic nanoparticles and gold nanoparticles. AFB1 is efficiently identified through an aptamer competition process facilitated by magnetic nanoparticles , which triggers multiple isothermal amplification. This converts trace amounts of the toxin into a large quantity of DNA signal. Upon specific activation of the CRISPR-Cas14a complex, the MAPs are cleaved, resulting in significant changes in both color and colorimetric signal. The method demonstrates acceptable sensitivity, with a detection limit of 31.90 pg mL-1 and a wide detection range from 0.05 to 10 ng mL-1. Furthermore, the assay exhibits satisfactory specificity and high accuracy when it is applied to practical samples. Our approach offers a universal sensing platform with potential applications in food safety, environmental monitoring, and clinical diagnostics.

A universal CRISPR-Cas14a responsive triple-sensitized upconversion photoelectrochemical sensor.

It has recently been discovered that, like other members of the Cas family (12a and 13a), the clustered regularly interspaced short palindrome repeat CRISPR-Cas14a system not only mediates high-sensitivity detection with exceptionally strong gene editing ability but is also generally useful for DNA detection via fluorescence. Photoelectrochemical (PEC) sensors have been widely applied as efficient analytical tools. Measuring electrical signals is more cost-effective and the necessary equipment is more easily portable than fluorescence signal detectors, but their stability still needs to be improved. The high base resolution of CRISPR-Cas14a can compensate for such shortcomings. Therefore, electrical signals and fluorescence signals were combined, and the development of a universal CRISPR-Cas14a-responsive ultrasensitive upconversion PEC sensor is described in this paper. Moreover, strand displacement amplification (SDA) and a near-infrared (NIR) light source were utilized to further improve the stability and sensitivity of the photoelectric signals. At the same time, the modified working electrode (UCNPs-ssDNA-CdS@Au/ITO) on the three-electrode disposable sensor was used as the reporter probe, which cooperates with the trans-cleavage activity of Cas14a endonuclease. To verify the universality of this sensor, the UCNPs-Cas14a-based PEC sensor was applied for the detection of the small-molecule toxin T2 and protein kinase PTK7. Here, we report that the limit of detection of this reagent was within the fg range, successfully applied to the detection of T2 in oats and PTK7 in human serum. We propose that by combining PEC and CRISPR-14a, UCNPs-Cas14a-based PEC sensors could become powerful drivers for the extensive development of ultrasensitive, accurate and cost-effective universal sensors for detection and diagnosis.

Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are widely used as gene editing tools in biology, microbiology, and other fields. CRISPR is composed of highly conserved repetitive sequences and spacer sequences in tandem. The spacer sequence has homology with foreign nucleic acids such as viruses and plasmids; Cas effector proteins have endonucleases, and become a hotspot in the field of molecular diagnosis because they recognize and cut specific DNA or RNA sequences. Researchers have developed many diagnostic platforms with high sensitivity, high specificity, and low cost by using Cas proteins (Cas9, Cas12, Cas13, Cas14, etc.) in combination with signal amplification and transformation technologies (fluorescence method, lateral flow technology, etc.), providing a new way for rapid detection of pathogen nucleic acid. This paper introduces the biological mechanism and classification of CRISPR-Cas technology, summarizes the existing rapid detection technology for pathogen nucleic acid based on the trans cleavage activity of Cas, describes its characteristics, functions, and application scenarios, and prospects the future application of this technology.

Comparative Analysis and Phylogenetic Insights of Cas14-Homology Proteins in Bacteria and Archaea.

Type-V-F Cas12f proteins, also known as Cas14, have drawn significant interest within the diverse CRISPR-Cas nucleases due to their compact size. This study involves analyzing and comparing Cas14-homology proteins in prokaryotic genomes through mining, sequence comparisons, a phylogenetic analysis, and an array/repeat analysis. In our analysis, we identified and mined a total of 93 Cas14-homology proteins that ranged in size from 344 aa to 843 aa. The majority of the Cas14-homology proteins discovered in this analysis were found within the Firmicutes group, which contained 37 species, representing 42% of all the Cas14-homology proteins identified. In archaea, the DPANN group had the highest number of species containing Cas14-homology proteins, a total of three species. The phylogenetic analysis results demonstrate the division of Cas14-homology proteins into three clades: Cas14-A, Cas14-B, and Cas14-U. Extensive similarity was observed at the C-terminal end (CTD) through a domain comparison of the three clades, suggesting a potentially shared mechanism of action due to the presence of cutting domains in that region. Additionally, a sequence similarity analysis of all the identified Cas14 sequences indicated a low level of similarity (18%) between the protein variants. The analysis of repeats/arrays in the extended nucleotide sequences of the identified Cas14-homology proteins highlighted that 44 out of the total mined proteins possessed CRISPR-associated repeats, with 20 of them being specific to Cas14. Our study contributes to the increased understanding of Cas14 proteins across prokaryotic genomes. These homologous proteins have the potential for future applications in the mining and engineering of Cas14 proteins.

Ligation-dependent Cas14a1-Activated biosensor for one-pot pathogen diagnostic.

Pathogen identification requires nucleic acid diagnosis with simple equipment and fast manipulation. Our work established an all-in-one strategy assay with excellent sensitivity and high specificity, Transcription-Amplified Cas14a1-Activated Signal Biosensor (TACAS), for the fluorescence-based bacterial RNA detection. The DNA as a promoter probe and a reporter probe directly ligated via SplintR ligase once specifically hybridized to the single-stranded target RNA sequence, with the ligation product transcribed into Cas14a1 RNA activators by T7 RNA polymerase. This forming sustained isothermal one-pot ligation-transcription cascade produced RNA activators constantly and enabled Cas14a1/sgRNA complex to generate fluorescence signal, thus leading to a sensitive detection limit of 1.52 CFU mL-1E. coli within 2 h of incubation time. TACAS was applied in contrived E. coli infected fish and milk samples, and a significant signal differentiation between positive (infected) and negative (uninfected) samples was reached. Meanwhile, E. coli colonization and transmit time in vivo were explored and the TACAS assay promoted the understanding of the infection mechanisms of the E. coli infection, demonstrating an excellent detection capability.

A signal-off Cas14a1-based platform for highly specific detection of methicillin-resistant Staphylococcus aureus.

Antibiotic usage has become very widespread in aquaculture, and the abuse or overuse of antibiotics has led to the evolution of antibiotic-resistance bacteria, which has adverse effects on aquatic products and ecosystems. Moreover, this evolution can potentially cause harm to human health. Thus, there is an urgent need for diagnostic tools for antibiotic-resistant microorganisms. Herein, we proposed a signal-off Cas14a1-based platform (SOCP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In this SOCP, we have designed single-stranded DNA (ssDNA) that not only can activate the trans-cleavage ability of dual Cas14a1-sgRNA complex but also can be used as the primers for the amplified methicilin-resistant gene (mecA). When MRSA is present, the primers can be transformed into products with amplification, leading to the signal decrease of trans-cleavage activity of Cas14a1. The SOCP showed high specificity and fair sensitivity for mecA gene and MRSA. In the detection of real samples, this platform also showed consistent results compared with qPCR. The SOCP could provide an alternative tool for the diagnosis of antibiotic-resistant bacteria in aquaculture, food industry and other fields.

Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets.

CRISPR-Cas systems have been widely used in genome editing and transcriptional regulation. Recently, CRISPR-Cas effectors are adopted for biosensor construction due to its adjustable properties, such as simplicity of design, easy operation, collateral cleavage activity, and high biocompatibility. Aptamers' excellent sensitivity, specificity, in vitro synthesis, base-pairing, labeling, modification, and programmability has made them an attractive molecular recognition element for inclusion in CRISPR-Cas systems. Here, we review current advances in aptamer-based CRISPR-Cas sensors. We briefly discuss aptamers and the knowledge of Cas effector proteins, crRNA, reporter probes, analytes, and applications of target-specific aptamers. Next, we provide fabrication strategies, molecular binding, and detection using fluorescence, electrochemical, colorimetric, nanomaterials, Rayleigh, and Raman scattering. The application of CRISPR-Cas systems in aptamer-based sensing of a wide range of biomarkers (disease and pathogens) and toxic contaminants is growing. This review provides an update and offers novel insights into developing CRISPR-Cas-based sensors using ssDNA aptamers with high efficiency and specificity for point-of-care setting diagnostics.

A novel CRISPR/Cas14a-based electrochemical biosensor for ultrasensitive detection of Burkholderia pseudomallei with PtPd@PCN-224 nanoenzymes for signal amplification.

In this work, PtPd nanoparticles functionalized porphyrin metal-organic framework nanoenzymes (PtPd@PCN-224 nanoenzymes) are exploited as signal amplification tags to fabricate a rapid and ultrasensitive sensitive CRISPR/Cas14a-based electrochemical biosensor for Burkholderia pseudomallei (B. pseudomallei) specific DNA sequences detection. The prepared PtPd@PCN-224 nanoenzymes not only catalyze the reduction peak current of H2O2 to obtain a strong electrochemical signal output, but also provide massive active sites for the assembly of nucleic acids by Zr-O-P bonds. Besides, the designed target-activated CRISPR/Cas14a is able to recognize the target DNA sequences and further trigger the trans-cleavage of ssDNA for signal amplification. Benefiting from the target-activated CRISPR/Cas14a and PtPd@PCN-224 nanoenzymes, the developed electrochemical biosensor for B. pseudomallei DNA detection exhibits high sensitivity with detection of limit down to 12.8 aM and excellent specificity for distinguishing non-targeted bacteria. Moreover, the CRISPR/Cas14a-based electrochemical detection platform can also apply for other pathogenic bacteria diagnostic by well-designing sgRNA for target sequence recognition, possessing high flexibility and versatility in clinical diagnosis.

CRISPR/Cas14 provides a promising platform in facile and versatile aptasensing with improved sensitivity.

CRISPR is reshaping biosensing technology due to its programmability, sensitivity, and specificity. Most current CRISPR-based biosensors are developed based on Cas12 and Cas13, while the biosensing potentials of the newly discovered Cas14 have not been fully elucidated yet. Herein, a fluorometric biosensor named HARRY (highly sensitive aptamer-regulated Cas14 R-loop for bioanalysis) was developed. The diblock ssDNA is designed to contain the activator sequence of Cas14 and the aptamer sequence of specific targets. In the absence of targets, the ssDNA activates Cas14a, then the Cas14a trans-cleavages the fluorescent reporter, causing fluorescence enhancement. In the presence of the targets, ssDNA-target assembly is formed via aptamer interaction, resulting in the inhibition of Cas14a activation. HARRY can detect ATP, Cd2+, histamine, aflatoxin B1, and thrombin with detection limits at the low-nanomolar level, which shows improvement compared with Cas12a-based aptasensors in sensitivity and versatility. We reasoned that the improvement is derived from the ssDNA specificity of Cas14a and found that the detection limit of HARRY is correlated to the binding affinities of aptamers. This study unlocks the potential of Cas14a in versatile aptasensing, which may inspire the development of CRISPR-based biosensors from the Cas14a branch.

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry.

The rapid rate of virus transmission and pathogen mutation and evolution highlight the necessity for innovative approaches to the diagnosis and prevention of infectious diseases. Traditional technologies for pathogen detection, mostly PCR-based, involve costly/advanced equipment and skilled personnel and are therefore not feasible in resource-limited areas. Over the years, many promising methods based on clustered regularly interspaced short palindromic repeats and the associated protein systems (CRISPR/Cas), i.e., orthologues of Cas9, Cas12, Cas13 and Cas14, have been reported for nucleic acid detection. CRISPR/Cas effectors can provide one-tube reaction systems, amplification-free strategies, simultaneous multiplex pathogen detection, visual colorimetric detection, and quantitative identification as alternatives to quantitative PCR (qPCR). This review summarizes the current development of CRISPR/Cas-mediated molecular diagnostics, as well as their design software and readout methods, highlighting technical improvements for integrating CRISPR/Cas technologies into on-site applications. It further highlights recent applications of CRISPR/Cas-based nucleic acid detection in livestock industry, including emerging infectious diseases, authenticity and composition of meat/milk products, as well as sex determination of early embryos.

Cas14a1-Mediated Nucleic Acid Diagnostics for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is the main genetic cause of infant death. In >95% of the patients with SMA, the disease is caused by a single hotspot pathogenic mutation: homozygous deletion of exon 7 of the survival motor neuron 1 gene (SMN1). Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-based assays have been developed as a promising new option for nucleic acid detection. Here, we developed a Cas14a1-based assay combined with asymmetric PCR to establish a method for detection of the homozygous deletion of SMN1 exon 7 in SMA patients. The minimum detectable concentration of genomic DNA reached 5.26 aM with our method, and the assessment of its detection performance in 33 clinical samples revealed that the results were completely consistent with those of multiple ligation-dependent probe amplification and quantitative PCR. Thus, our novel nucleic acid diagnostics combining CRISPR/Cas14a1 and asymmetric PCR not only provides specific and sensitive testing of the deletion of SMN1 exon 7, but also holds promise for an accurate detection platform of genetic diseases and pathogens in multiple sample types.

CRISPR-Cas14a-integrated strand displacement amplification for rapid and isothermal detection of cholangiocarcinoma associated circulating microRNAs.

Circulating microRNAs (miRNA) can serve as key biomarkers for early diagnose of cholangiocarcinoma. Herein, an assay that uses circulating miRNA to trigger strand displacement amplification (SDA) and a CRISPR-Cas14a system to report the SDA process has been developed. In the proposed method, SDA directly amplifies miRNAs without reverse transcription. The reporter, CRISPR-Cas14a, can reduce the risks of non-specific amplification and offers a sequential amplification that improves the sensitivity for miRNA detection. The assay, termed Cas14SDA, can discriminate miRNAs with similar sequences and can detect as low as 680 fM miR-21 (miRNAs overexpressed in cholangiocarcinoma) within 1 h. In particular, Cas14a was efficiently activated by a single-stranded SDA amplicon which improved the sensitivity by 2.86 times compared to that using Cas12a. This research has demonstrated that the Cas14SDA assay can discriminate cholangiocarcinoma patients from healthy donors by testing miR-21 in their blood samples. The Cas14SDA assay developed broadens the toolbox for miRNA biomarker analysis.

A novel CRISPR/Cas14a system integrated with 2D porphyrin metal-organic framework for microcystin-LR determination through a homogeneous competitive reaction.

Selective and sensitive detection of microcystin-LR (MC-LR) is of vital importance because of its high toxicity and broad distribution. Herein, a novel and versatile fluorescence sensor (Cas14-pMOFs fluorescence sensor) was developed by combining the CRISPR/Cas14a system with a 2D porphyrin metal-organic framework nanosheets (2D-pMOFs) for MC-LR determination. The designed CRISPR/Cas14a system was activated by the unbound complementary DNA (cDNA), which was positively correlated with MC-LR concentration. Furthermore, the activated Cas14a protein was utilized to indiscriminately cleave the FAM-labeled single-stranded DNA (ssDNA-FAM), which was pre-absorbed on Cu-TCPP(Fe) nanosheets. Because of the desorption of the cleaved ssDNA-FAM, the pre-quenched fluorescence signal was recovered. Owing to the excellent performance in quantifying cDNA using this Cas14-pMOFs fluorescence sensor with a limit of detection (LOD) of 0.12 nM, this Cas14-pMOFs fluorescence sensor was able to detect MC-LR in a range from 50 pg/mL to 1 µg/mL with the LOD of 19 pg/mL. This work not only provided a new insight for the exploration of fluorescence sensors based on 2D-pMOFs coupled with CRISPR/Cas14a, but also, demonstrated its universality in both nucleic acid and non-nucleic acid targets determination.

trans Single-Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction.

As a CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here, we describe an unreported capacity of Cas14a1: RNA can trigger the trans ssDNA cleavage. This Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNA cleavage, ATCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNA-activation. Using ATCas-RNA via a fluorophore quencher-labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100 % accuracy with 25 milk samples. This platform can serve as a new tool for high-efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.

Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato.

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I-IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.