Cell engineering is commonly limited to the serial manipulation of a single gene or locus. The recently discovered CRISPR-associated transposases (CASTs) could manipulate multiple sets of genes to achieve predetermined cell diversity, with orthogonal CASTs being able to manipulate them in parallel. Here, a novel CAST from Pseudoalteromonas translucida KMM520 (PtrCAST) was characterized without a protospacer adjacent motif (PAM) preference which can achieve a high insertion efficiency for larger cargo and multiplexed transposition and tolerate mismatches out of 4-nucleotide seed sequence. More importantly, PtrCAST operates orthogonally with CAST from Vibrio cholerae Tn6677 (VchCAST), though both belonging to type I-F3. The two CASTs were exclusively active on their respective mini-Tn substrate with their respective crRNAs that target the corresponding 5 and 2 loci in one Escherichia coli cell. The multiplexed orthogonal MUCICAT (MUlticopy Chromosomal Integration using CRISPR-Associated Transposases) is a powerful tool for cell programming and appears promising with applications in synthetic biology.

/pdf/orthogonal-crispr-associated-transposases-for-parallel-and-54oy6a738p.pdf

Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration.

Despite significant advancements in medical technology, cancer remains the world's second‐leading cause of death, largely attributed to late‐stage diagnoses. While traditional cancer detection methodologies offer foundational insights, they often lack the specificity, affordability, and sensitivity for early‐stage identification. In this context, the development of biosensors offers a distinct possibility for the precise and rapid identification of cancer biomarkers. Carbon nanomaterials, including graphene, carbon nitride, carbon quantum dots, and other carbon‐based nanostructures, are highly promising for cancer detection. Their simplicity, high sensitivity, and cost‐effectiveness contribute to their potential in this field. This review aims to elucidate the potential of emerging carbon‐nanomaterial‐based biosensors for early cancer diagnosis. The relevance of the various biosensor mechanisms and their performance to the physicochemical properties of carbon nanomaterials is discussed in depth, focusing on demonstrating broad methodologies for creating performance biosensors. Diverse carbon‐nanomaterial‐based detection techniques, such as electrochemical, fluorescence, surface plasmon resonance, electrochemiluminescence, and quartz crystal microbalance, are emphasized for early cancer detection. At last, a summary of existing challenges and future outlook in this promising field is elaborated.

Emerging Multifunctional Carbon‐Nanomaterial‐Based Biosensors for Cancer Diagnosis

Recent advances in phage defense systems and potential overcoming strategies.

The physiological and clinical importance of Glutathione and Cysteamine is emphasized by their participation in a range of conditions, such as diabetes, cancer, renal failure, Parkinson's disease, and hypothyroidism. This necessitates the requirement for accessible, expedited, and cost-efficient testing that can facilitate clinical diagnosis and treatment options. This article examines numerous techniques used to detect both glutathione and cysteamine. The discussed methods include electroanalytical techniques such as voltammetry and amperometry, which are examined for their sensitivity and ability to provide real-time analysis. Furthermore, this study investigates the accuracy of gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) in measuring the concentrations of glutathione and cysteamine. Additionally, the potential of new nanotechnology-based methods, such as plasmonic nanoparticles and quantum dots, to improve the sensitivity of detecting glutathione and cysteamine is emphasized. 

Unveiling thiol biomarkers: Glutathione and cysteamine

Glutathione (GSH) is a crucial non-protein thiol and an indispensable endogenous antioxidant. The aberrant expression of GSH in plasma and cytosol is closely related to numerous diseases, including cancer. Therefore, establishing a sensitive method for analyzing GSH has important application value for biomedical research and clinical medical detection. Herein, A method for the rapid and simple detection of GSH was proposed, which is based on an anti-etching mechanism by utilizing gold nanotetrapods (Au NTPs) and manganese dioxide nanosheets (MnO2 NSs). In the absence of GSH, Au NTPs solution can cause a distinct color change from gray-green to red through the etching effect of MnO2 NSs. However, in the presence of GSH, the redox reaction between GSH and MnO2 NSs inhibits the etching of Au NTPs by MnO2 NSs, and Au NTPs solution maintains persistent gray-green color. The colorimetric probe exhibited excellent selectivity for GSH. The limits of detection for GSH were 43.5 nM (UV–vis spectrum) and 0.25 μM (naked eyes). The sensing technique exhibited excellent linearity between wavelength shift and GSH concentration within the range of 0.25 μM–1.5 μM. The outcomes of GSH detection in actual biological samples demonstrate that this probe has the potential to be applied to GSH detection in intricate biological samples. 

Multicolor detection of glutathione by manganese dioxide nanosheets and gold nanotetrapods based on an anti-etching mechanism

Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5′ phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3′ of the target RNA which is recognized by 5′ sequence of the gDNA. These results confirmed that NgAgo–gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3′ of the target RNA, which is a new finding that has not been reported in the past.

Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets

Construction of continuously enhanced fluorescent sensor for detection of glutathione in normal and cancer cells.

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Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets

Construction of continuously enhanced fluorescent sensor for detection of glutathione in normal and cancer cells.