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At least 19 recordsLinked to original sources
PubMed2026

Engineering CRISPR for Point-of-Care Tests.

CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.

CRISPR-Cas Systems↗
PubMed2026

Structure and evolution-guided design of minimal RNA-guided nucleases.

The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.

Humans↗
PubMed2026

Inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production.

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation or gravity sedimentation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY® VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

Flocculation↗
PubMed1999

Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST.

The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130(Cas) as a substrate, both in vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130(Cas) than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130(Cas) is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130(Cas) to the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130(Cas) with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130(Cas), thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130(Cas) and regulating cell migration.

Animals↗
PubMed2025

Integration of therapeutic cargo into the human genome with programmable type V-K CAST.

CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.

Humans↗
PubMed2025

Charting the development and engineering of CRISPR base editors: lessons and inspirations.

CRISPR base editors (BEs) have introduced a new chapter in precise genome editing. The brief but fruitful history of BE development documents many case studies that not only lay the foundation of base-editing technology but are also instrumental to future protein engineering efforts. In this review, we summarize the development and engineering of various BEs with a focus on recent progress. These include traditional cytosine and adenine base editors (CBEs and ABEs), novel TadA-derived CBEs, transversion BEs, dual BEs, and CRISPR-free BEs. We discuss each aspect of the workflow and highlight the successes and challenges encountered in the engineering process.

Gene Editing↗
PubMed2006

Structural basis for the nuclear protein import cycle.

Transport of macromolecules between the nuclear and cytoplasmic compartments through NPCs (nuclear pore complexes) is mediated by soluble transport factors that are commonly members of the importin-beta superfamily. In the nuclear protein import cycle, importin-beta binds cargo in the cytoplasm (usually via the importin-alpha adaptor) and transports it through NPCs with which it interacts transiently by way of NPC proteins ('nucleoporins') that contain distinctive FG (Phe-Gly) sequence repeats. In the nucleus, Ran-GTP binds to importin-beta, dissociating the import complex. The importin-beta-Ran-GTP complex recycles to the cytoplasm, whereas importin-alpha is recycled by the importin-beta family member CAS (cellular apoptosis susceptibility protein) complexed with Ran-GTP. Cytoplasmic RanGAP (Ran GTPase-activating protein) dissociates these complexes, freeing the importins for another import cycle. Crystallography and biochemical and cellular studies have enabled a molecular description of the transport cycle to be developed and tested using protein engineering and computer modelling. Importin-beta family members are elongated flexible molecules that adapt their shape to encircle their cargoes. Ran-GTP binds at three sites along importin-beta and CAS, locking the molecules into a rigid conformation that is unable to bind cargoes effectively. Interactions between transport factors and key nucleoporins (such as Nup1p, Nup2p and Nup50) accelerate the formation and dissolution of many of these complexes.

Animals↗
PubMed2026

Miniature and versatile genome regulation TnpB-ωRNA toolkits facilitate cancer immunotherapy.

CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy.

Humans↗
PubMed2023

Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

Escherichia coli↗
PubMed2025

Bacterial directed evolution of CRISPR base editors.

Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.

Gene Editing↗
PubMed2025

Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.

CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.

Cryoelectron Microscopy↗
PubMed2026

Active-site arginines differentially control Cas12a DNA cleavage and specificity.

Cas12a is a CRISPR-Cas nuclease with biochemical features that make it useful for genome editing and nucleic acid diagnostics. However, its off-target and non-specific trans and CRISPR RNA-independent DNA cleavages can reduce the accuracy and limit applications requiring high fidelity. Here, we analyzed the role of two conserved arginine residues, R918 and R921, found in the RuvC active site pocket of Francisella novicida Cas12a. Through amino acid substitutions, biochemical assays, kinetic analysis, and computational study, we establish that a positive charge at 921 is required for CRISPR RNA-dependent DNA cleavage (cis cleavage), whereas R918 primarily enhances cleavage efficiency. Replacing R918 with lysine or alanine eliminates trans activity while retaining cis cleavage, whereas replacing R921 with lysine eliminates trans activity and replacing with alanine abolishes cis and trans cleavages. Furthermore, these changes significantly decrease RNA-independent cleavage and improve mismatch discrimination during cis cleavage, especially at PAM-distal sites. Structural analysis shows that R918 assists in the conversion of the lid covering the RuvC active site to an alpha helical form, while R921 stabilizes the DNA in the active site. Molecular dynamics simulations reveal that while R921 is critical in supporting the positioning of scissile phosphate, R918 is essential in maintaining catalytic-site organization through lid's conformational change as well as in positioning DNA through its role in stabilizing the active site framework. Together, our results highlight the importance of R918 and R921 in Cas12a's activity and the potential of modifying active pocket residues to reduce unwanted DNA cleavage while increasing on-target specificity.

CRISPR-Cas↗
PubMed2026

Stepwise DNA-unwinding gates TnpB genome-editing activity.

TnpB is a compact RNA-guided endonuclease and an evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited, and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived, partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, H4W-L304F-V305R (Ymu1-WFR), stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

Gene Editing↗
PubMed2026

Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.

CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.

Animals↗
PubMed2026

Repeat region engineering of Cas13a crRNA enables conformational gating-based autocatalytic CRISPR biosensing.

CrRNA engineering has emerged as a pivotal strategy for extending CRISPR-Cas13a biosensing. However, structural modulation of the direct repeat (DR) region remains exceptionally challenging due to its intricate architecture and the high energetic barrier of the Cas13a-crRNA interface, which is conventionally viewed as a rigid and immutable scaffold. Here, we demonstrate that the DR region is instead a programmable topological element with unexpected structural plasticity. By systematically engineering the DR through sequence insertion and structural splitting, we identified multiple DR variants that retain robust catalytic activity. Crucially, this topological reconfiguration enables Cas13a activity to be precisely gated by unmodified nucleic acid blockers, a level of regulation unattainable with the wild-type crRNA. Building on this flexible modulation, we developed Dre-CRISPR, a DR-engineered platform that couples target-triggered DR restoration to a self-reinforcing autocatalytic loop. This self-amplifying system provides a 2 × 106-fold sensitivity enhancement over nonamplified systems. Furthermore, the Dre-CRISPR platform extends the diagnostic scope of Cas13a to a broader spectrum of analytes, ranging from microRNAs to enzymatic activities and heavy metal ions. Our findings redefine the crRNA scaffold as a versatile signaling node and provide a generalizable framework for developing high-sensitivity, self-amplifying CRISPR biosensors through topology-driven guide RNA engineering.

CRISPR-Associated Proteins↗
PubMed2000

Cloning and expression of functional equistatin.

Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D. In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina, was screened. A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence. The mature protein region was amplified by PCR, cloned into the pET22b(+)cas expression vector and expressed in Escherichia coli. Isolation of active recombinant equistatin required only one purification step, the His-tag affinity column. The protein displays physical and inhibitory properties closely similar to the native inhibitor.

Amino Acid Sequence↗
PubMed2025

Re-arranging the Cis-regulatory Modules of Hox Complex in Drosophila via FLP-FRT and CRISPR/Cas9.

FLP-FRT, a well-established technique for genome manipulation, and the revolutionary CRISPR/Cas9, known for its targeted indels, are combined in a novel approach. This unique method is applied to the Hox genes in the Drosophila melanogaster bithorax complex, which are closely located to the cis-regulatory modules that define their spatial-temporal regulation. The number and position of these genes are directly correlated to their expression pattern. This chapter unveils the exciting potential of this combinatorial use of FLP-FRT and CRISPR-Cas9 to rearrange the cis-regulatory modules of the Hox complex in Drosophila melanogaster.

Animals↗
PubMed2026

When Homing Endonuclease Meets Transposon: The OMEGA System.

Sequence-specific DNA endonucleases have made significant contributions to biology, biotechnology, and medicine; restriction enzymes and homing endonucleases are among classic examples. The demonstration of programmable genome editing using Cas9 in the CRISPR-Cas system, in which the target DNA sequence is recognized by base pairing with a guide RNA, revolutionized the field of genome engineering, making target selection more flexible and convenient. The OMEGA (Obligate Mobile Element-Guided Activity) system, considered a precursor to Cas12, and likely to Cas9, in the CRISPR-Cas system, is an RNA-guided DNA endonuclease composed of a TnpB, IscB, IsrB, or Fanzor protein, and a structural RNA designated reRNA or ωRNA. The OMEGA system is present in the three domains of life as an auxiliary component of transposons. The OMEGA system cuts DNA in an allele from which a transposon is excised and triggers recombination to reinstate the transposon at the same position. This "transposon restorative homing" redefines the OMEGA system as a homing endonuclease. In this review, the selfish aspects of the OMEGA system are discussed in the historical context of homing endonuclease research.

Cas12↗