Chimeric antigen receptor (CAR)-T cells exhibit low antigen sensitivity, which restricts their therapeutic efficacy and leads to patient relapses when cancer cells downregulate antigen expression. Despite the pressing need to overcome this limitation, the underlying mechanisms remain poorly understood. Here, we demonstrate that enhancing CAR sensitivity to match the sensitivity of the T-cell receptor (TCR) can be achieved by engineering matched extracellular sizes of CAR/antigen and CD2/CD58 complexes. We find that different CAR/antigen sizes, which are generated by different CAR architectures and different target antigens, require a different CD2/CD58 size to optimise sensitivity. This extracellular size-matching improves antigen engagement and co-localisation of CAR/antigen and CD2/CD58 complexes. We also find that size-matching controls co-inhibition of CARs by PD-1/PD-L1. These findngs highlight the importance of size-matching for signal integration by surface receptors and offers a new approach to tune CAR-T cell sensitivity by matching or mismatching extracellular sizes. One sentence summary The antigen sensitivity of CAR-T cells can be tuned to match the sensitivity of TCR-T cells by varying the relative extracellular size of CAR/antigen and CD2/CD58 complexes. Graphical abstract

CRISPR-Cas systems often rely on collateral cleavage activities against nucleic-acid substrates to combat recognized mobile genetic elements. Of the associated RNA-guided Cas effector nucleases, Cas12a2 stands out as the only known example exhibiting RNA-triggered collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA. However, little is known about the underlying mechanisms of collateral cleavage, including which of these activities dominates during the Cas12a2-mediated immune response. Here, we show that Cas12a2 cleaves collateral DNA substrates over RNA substrates, even when RNA substrates are more abundant. This preference relies on a positive cooperativity mechanism that requires the four “aromatic clamp” residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while the RNA-targeting Cas13a cannot. This work thus reveals how Cas12a2 decides amongst different collateral cleavage substrates, with immediate implications for understanding Cas12a2-based immunity, improving molecular diagnostics, and laying a mechnastic foundation for future Cas12a2-based technologies.

Cryptococcus neoformans infections are a significant cause of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. The cryptococcal cell wall is very dynamic and can be modulated depending on growth conditions. It was reported that when C. neoformans is grown in unbuffered yeast nitrogen base (YNB) for 48 hours, the pH of the media drastically drops and the cells start to shed their cell walls. With this observation, we sought to determine if YNB-grown cells could be used directly for genetic transformation. To test this, we targeted ADE2 using TRACE (transient CRISPR–Cas9 coupled with electroporation) in YNB-grown or competent cells. Deletion of the ADE2 gene results in red-pigmented colonies allowing visual confirmation of disruption. We were able to successfully delete ADE2 in YNB-grown cells and with better efficiency compared to competent cells. Recently, it was shown that gene deletion can be accomplished using short (50 bp) homology arms in place of the normal long arms (∼1 kb). However, it was inefficient, leading to more insertions and gene disruption than gene deletions. We tested short homology with YNB-grown cells vs. competent cells and found gene deletion was significantly improved in YNB-grown cells, around 60%, compared to around 6% with the competent cells. This was also observed when we deleted LAC1 with the short arms. Altogether, using simple growth conditions, we have greatly improved the speed and efficiency of cryptococcal genetic transformations. Importance The World Health Organization recently ranked C. neoformans as the #1 highest-priority fungal pathogen based on unmet research and development needs and public health importance. Understanding cryptococcal pathogenicity is key for developing treatments. We found that using simple growth conditions can greatly improve the speed and efficiency of cryptococcal genetic transformations. This finding will advance the field by expanding the ease of cryptococcal genetic manipulations.

X-chromosome inactivation (XCI) is initiated during early mammalian embryogenesis by a long non-coding RNA (lncRNA) XIST , which coats one of the two X-chromosomes and facilitates epigenetic transcriptional silencing. A second, evolutionarily recent primate-specific lncRNA XACT was proposed to antagonize XIST ’s ability to induce XCI. XACT expression is restricted to pluripotent states and early embryonic stages and coats the active X-chromosome in both females and males. Here, we report a novel XACT transcript expressed in normal and cancerous somatic cells from both the inactive (Xi) and active (Xa) X chromosomes. It coexists with XIST on the Xi without affecting XIST expression or (re)activating X-linked genes inactivated by XCI. During hematopoietic stem cell (HSC) differentiation, XACT is primarily expressed in myeloid progenitors in both sexes. XACT expression is activated in HSCs and peaks in megakaryocyte-erythrocyte progenitor cells (MEPs) before rapidly declining as the MEPs differentiate into megakaryocytes or erythrocytes. By combining CRISPR-based XACT perturbation with epigenomic and transcriptional studies, we revealed the key role of XACT in the self-renewal and differentiation of erythroid progenitors into erythrocytes, by recruiting cis-regulatory proteins and regulating transcription through ETS and AP-1 transcription factors. Furthermore, XACT is expressed in a subset of acute myeloid leukemia (AML) patients, with high levels found in erythroid-megakaryocytic blast cells; suggesting XACT ’s potential as a marker for AML subtypes. Thus, we identified a novel XACT transcript with a unique expression profile and important roles in normal and cancer cells, revealing XACT lncRNA functions beyond its previously proposed role in XCI during embryogenesis.

Psychrophilic bacteria, also known as cold-loving bacteria, are microorganisms that thrive in extremely cold environments, typically at temperatures ranging from -5°C to 15°C. These bacteria are found in habitats such as the deep ocean, polar regions, and high-altitude environments. Because of their unique ability to survive and function at low temperatures, psychrophilic bacteria are becoming increasingly important in bioengineering and biotechnology. Their potential and future in bioengineering include enzyme production for cold-temperature industrial applications (bioremediation, food industry, pharmaceuticals and diagnostics), biocatalysis in cold-chain biotechnology, bioengineering cold-tolerant crops and organisms, cold-adapted microbial consortia for bioremediation, astrobiology, synthetic biology, and development of novel CRISPR-Cas proteins. As we continue to better understand and harness the genetic and biochemical properties of these bacteria, we may unlock even more possibilities for the future of biotechnology and bioengineering.

Autophagy, a critical process for the vacuolar degradation of proteins and organelles, is governed by multiple conserved autophagy-related (ATG) proteins. The central component of the ATG machinery is the ubiquitin-like protein ATG8, which is essential for multiple steps of the autophagy process, including phagophore expansion, autophagosome closure, trafficking and fusion with the lysosome/vacuole, and selective cargo recruitment. Currently, our understanding of the roles of ATG8 in plant autophagy and the functional specialization of ATG8 family members is limited due to genetic redundancy. To assess the roles of ATG8 genes in plant autophagy, here we used CRISPR/Cas9 technology to systematically knockout the Arabidopsis ATG8 genes. By analyzing the atg8 mutants, we found that in contrast to mammalian ATG8s, in which the LC3s and GABARAP subfamilies play distinct roles in the autophagic process, Arabidopsis ATG8s perform an overlapping function in controlling autophagic flux. Combinatorial mutations of Clade I and Clade II ATG8s resulted in severely impaired autophagy under nutrient-starved conditions. Furthermore, we found that RABG3 proteins, members of the RAB7/RABG GTPase family, interact with ATG8s through AIM-LDS interfaces, and that such interaction is essential for the association of RABG3 proteins with the autophagosomal membrane and probably for the fusion of autophagosome with the vacuole, but is not required for endosomal trafficking. With the collection of multiple high-order atg8 mutants generated in this study, we now provide a venue to study the roles of ATG8 genes in canonical autophagy and non-canonical autophagy in Arabidopsis .

A20, encoded by the TNFAIP3 gene, is a protein linked to Crohn’s disease and celiac disease in humans. We now find that mice expressing point mutations in A20’s M1 ubiquitin binding motif (ZF7) spontaneously develop proximate enteritis that requires both luminal microbes and T cells. Cellular and transcriptomic profiling reveal expansion of TH17/22 cells and aberrant expression of IL-17A and IL-22 in intestinal lamina propria of A20 ZF7 mice. While deletion of IL-17A from A20 ZF7/ZF7 mice exacerbates enteritis, deletion of IL-22 abrogates intestinal epithelial cell hyperproliferation, barrier dysfunction, and alarmin expression. A20 ZF7/ZF7 TH17/22 cells autonomously express more RORψt and IL-22 after differentiation in vitro . ATAC sequencing identified an enhancer region upstream of the Il22 gene in A20 ZF7/ZF7 T cells, and this enhancer demonstrated increased activating histone acetylation coupled with exaggerated Il22 transcription. Finally, CRISPR/Cas9-mediated ablation of A20 ZF7 in human T cells increases RORψt expression and IL22 transcription. These studies link A20’s M1 ubiquitin binding function with RORψt expression, epigenetic activation of TH17/22 cells, and IL-22 driven enteritis.

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating muscle disease caused by de-repression of the toxic DUX4 gene in skeletal muscle. FSHD patients may benefit from DUX4 inhibition therapies, and although several experimental strategies to reduce DUX4 levels in skeletal muscle are being developed, no approved disease modifying therapies currently exist. We developed a CRISPR-Cas13b system that cleaves DUX4 mRNA and reduces DUX4 protein level, protects cells from DUX4-mediated death, and reduces FSHD-associated biomarkers in vitro . In vivo delivery of the CRISPR-Cas13b system with adeno-associated viral vectors reduced acute damage caused by high DUX4 levels in a mouse model of severe FSHD. However, protection was not sustained over time, with decreases in Cas13b and guide RNA levels between 8 weeks and 6 months after injection. In addition, wild-type mice injected with AAV6.Cas13b showed muscle inflammation with infiltrates containing Cas13b-responsive CD8+ cytotoxic T cells. Our RNA-seq data confirmed that several immune response pathways were significantly increased in human FSHD myoblasts transfected with Cas13b. Overall, our findings suggest that CRISPR-Cas13b is highly effective for DUX4 silencing but successful implementation of CRISPR/Cas13-based gene therapies may require strategies to mitigate immune responses.

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.

Summary Cyclic oligonucleotide-based antiviral signaling systems (CBASS) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 Å crystal structure of Acb4 in complex with 3′3′-cGAMP reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo . Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.

ABSTRACT RNA molecule plays an essential role in a wide range of biological processes. Gaining a deeper understanding of their functions can significantly advance our knowledge of life’s mechanisms and drive the development of drugs for various diseases. Recently, advances in RNA foundation models have enabled new approaches to RNA engineering, yet existing methods fall short in generating novel sequences with specific functions. Here, we introduce RNAGenesis, a foundation model that combines RNA sequence understanding and de novo design through latent diffusion. With a Bert-like Transformer encoder with Hybrid N-Gram tokenization for encoding, a Query Transformer for latent space compression, and an autoregressive decoder for sequence generation, RNAGenesis reconstructs RNA sequences from learned representations. Specifically for the generation, a score-based denoising diffusion model is trained to capture the latent distribution of RNA sequences. RNAGenesis outperforms current methods in RNA sequence understanding, achieving the best results in 9 of 13 benchmarks (especially in RNA structure prediction), and further excels in designing natural-like aptamers and optimized CRISPR sgRNAs with desirable properties. Our work establishes RNAGenesis as a powerful tool for RNA-based therapeutics and biotechnology.

The production of overwintering eggs is a critical adaptation for winter survival among many insects. Melanization contributes to eggshell pigmentation and hardening, consequently enhancing resistance to environmental stress. The complex life cycle of the pea aphid ( Acyrthosiphon pisum ), a model hemipteran insect with remarkable reproductive capacity, involves cyclical parthenogenesis. It enables the production of black overwintering eggs that undergo obligate diapause to survive under unfavorable conditions. Laccase2 ( Lac2 ) is essential for cuticle sclerotization and pigmentation in other insects. We hypothesized that Lac2 plays a critical role in aphid eggshell pigmentation and survival during diapause. To test the hypothesis, we used CRISPR/Cas9 ribonucleoprotein microinjections and a novel Direct Parental CRISPR (DIPA-CRISPR) method to knockout Lac2 . In Lac2 knockout (KO) crispants (G0), pigment-less eggs correlated with induced indel rates. Additionally, eggshell pigmentation was completely lost in homozygous Lac2 knockouts, leading to embryonic lethality. Observation of late-stage embryos in KO diapause eggs suggested that lethality occurred during late embryogenesis or hatching. Furthermore, eggshell stiffness was significantly reduced in Lac2 KOs, highlighting the role of this gene in eggshell hardening. Moreover, fungal growth was observed in KO eggs. These findings reveal the essential roles of Lac2 in eggshell pigmentation, hardening, late embryonic development, hatching, and fungal protection, which are critical for pea aphid survival during overwintering diapause. This study also advances CRISPR/Cas9-mediated genome editing in pea aphids by addressing the challenges associated with their unique biology, including complex life cycles, obligatory diapause, bacterial endosymbiosis, inbreeding depression, and high nuclease activity. Our optimized protocol achieved efficient targeted mutagenesis and germline transmission, thereby generating stable KO lines. Additionally, we successfully applied DIPA-CRISPR to aphids by inducing mutations via adult oviparous female injections in fertilized eggs. These robust genome-editing protocols will facilitate functional studies in aphids, a key model for research on evolution, ecology, development, and agriculture. Author Summary Surviving harsh winters is a challenge for many insects, and the production of specialized overwintering eggs is a common adaptation strategy. These eggs are protected from cold, desiccation, and fungal infections by their hardened, pigmented shells. The pea aphid, a hemipteran insect with a complex life cycle, relies on these eggs to survive winter. Aphids alternate between asexual reproduction in the warmer months and sexual reproduction in the fall, producing overwintering eggs that remain dormant until spring. In this study, we explored the role of Laccase2 ( Lac2 ), a key gene that contributes to the strengthening and darkening of insect exoskeletons. We disrupted Lac2 in pea aphids using advanced genome-editing techniques, including a new approach called DIPA-CRISPR. This resulted in pigment-free eggs with weakened shells that were more prone to fungal infections and failed to hatch. This demonstrates that Lac2 is essential for the survival of overwintering eggs. In addition, we refined the CRISPR/Cas9 genome editing methods for pea aphids, enabling efficient and precise genetic studies. These findings and the tools we developed can facilitate research on ecology, evolution, and pest control, while shedding light on how insects adapt to challenging environments.

CAR-T therapies utilizing T cells engineered with chimeric antigen receptors (CARs) have revolutionized the treatment of hematologic and immune-related malignancies. However, the anti-tumor capability of engineered CAR-T cells are often not persistent, which greatly dampen its clinical efficacy. To address this limitation, massive parallel genetic screens were widely used to identified novel targets and regulators that enhance T cell anti-tumor capability and persistence in tumor microenvironment. We hypothesized that by combining the pooled screen data from multiple independent genetic screens we could provide a systematic, comprehensive, and robust analysis of the effect of gene perturbation on T-cell based immunotherapies. After collecting data from previously published T cell screens, including CRISPR-based and ORF-based screens, through Gene Expression Omnibus (GEO), we reprocessed the gene hits summary and conducting a pathway enrichment analysis. A T cell screen perturbation score (TPS) was employed to quantifies the impact of the gene on T cell function. Additionally, gene expression data (both bulk RNA level and single-cell RNA level) from autoimmune disease and T cell-derived cancers were analyzed to pick up gene perturbations that potentially augment T cell proliferation. We integrated all data and analysis on 27 T-cells screens into our state-of-the-art T cell perturbation genomics database (TCPGdb), which is made easily accessible through our webserver and allows users to interactively explore the impact of query genes on T cell function based on prior screen data and our TPS scoring module. TCPGdb is publicly accessible at http://tcpgdb.sidichenlab.org/ .

African Swine Fever Virus (ASFV) is a high consequence, highly transmissible pathogen affecting swine causing African Swine Fever (ASF), a devastating disease, with high mortality rates in naive populations. Due to the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States (U.S.) to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten U.S. swine due to their proximity and involvement in movement to and from North America. While surveillance programs are ongoing, there are limited point-of-care (POC) tests available during outbreaks that maintain the sensitivity and specificity standards of laboratory testing (e.g., qPCR). However, the recently developed CRISPR-Cas testing systems may provide comparable high-quality results. In a CRISPR-based diagnostic assay, CRISPR effectors can be programmed with CRISPR-RNA (crRNA) to target specific DNA or RNA. Upon target binding, the Cas enzyme undergoes collateral cleavage of nearby fluorescently quenched reporter molecules (ssDNA or ssRNA), which can be detected under blue light or a fluorescence microplate reader. Furthermore, this tool is rapid, simple, cost-effective and can be performed with inexpensive equipment. For these reasons, we sought to develop a low-cost visual detection method for ASFV by employing the recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a point-of-care-assay. Our CRISPR-Cas12a assay demonstrated comparable sensitivity and specificity to qPCR, both visually and when quantified using a fluorescent reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, the CRISPR assay achieved a sensitivity of 98.3% (102 DNA copies) and a specificity of 100%. Finally, an assessment of the reaction time constraints indicated that results can be visualized in as little as seven minutes with a peak fluorescence at 40 min (RPA and CRISPR steps). The results of this feasibility assay validation allow for the rapid development of sensitive and specific POC tests that may be used for outbreak response in the future.

Cancer cells display distinct, recurrent phenotypic cell states. Metastatic spreading correlates with tumor cell state evolution. However, the molecular mechanisms underlying metastasis remain elusive. Here, we demonstrate that the quantitative dosage of oncogenic KRAS drives lung adenocarcinoma progression and metastasis via the integration of external signaling and pioneer transcription factor dynamics into qualitative cell states. Combining mouse models, in vivo CRISPR activation screens, and fate mapping, we show that even mild transcriptional amplification of KRAS significantly fuels tumor progression and metastasis. Chromatin profiling and transcriptomics reveal that high and low KRAS dosages supersede and integrate inflammatory and TGFβ signaling to dictate mouse cancer cell states. Patient data show that KRAS dosages correlate with distinct survival outcomes, transcription factor activity, and cell states. Direct KRAS inhibition in xenografts limits the KRAS-high “proliferative” cell state but spares a minimal residual state mimicking the KRAS-low “ciliated-like” state. Thus, oncogenic KRAS dosage fuels tumor heterogeneity at the cell state level and drives a bimodal tumor evolution during metastasis, with implications for prognosis and treatment.

During classical non-homologous end joining (cNHEJ), DNA-dependent protein kinase (DNA-PK) encapsulates free DNA ends, forming a recruitment platform for downstream end-joining factors including Ligase 4 (LIG4) 1 . DNA-PK can also bind telomeres and regulate their resection 2–4 , but does not initiate cNHEJ at this position. How the end joining process is regulated in this context-specific manner is currently unclear. Here we show that the shelterin components TRF2 and RAP1 form a complex with DNA-PK that directly represses its end joining function at telomeres. Biochemical experiments and cryo-electron microscopy reveal that when bound to TRF2, RAP1 establishes a network of interactions with KU and DNA that prevents DNA-PK from recruiting LIG4. In mouse and human cells, RAP1 is redundant with the Apollo nuclease in repressing cNHEJ at chromosome ends, demonstrating that the inhibition of DNA-PK prevents telomere fusions in parallel with overhang-dependent mechanisms. Our experiments show that the end joining function of DNA-PK is directly and specifically repressed at telomeres, establishing a molecular mechanism for how individual linear chromosomes are maintained in mammalian cells.

CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show “leaky” Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be distinctly problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we established transgenic platforms that minimize Cas9 functionality in the off-state along with maximized and uncompromised on-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (on vs. off-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we created a branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.

Acute myeloid leukaemia (AML) is often aggressive and life-threatening with limited curative options. Immunotherapies including chimeric antigen receptor (CAR) T cell approaches are under investigation, but high levels of disease heterogeneity remain a major hurdle to achieving durable responses. Targeting of multiple surface antigens may ensure complete immunological coverage of leukemic blast populations, but such antigens are often also present on healthy haematopoietic populations. To address likely aplasia, strategies can be designed to bridge CAR T cell therapies to allogeneic stem cell transplantation (allo-SCT), as demonstrated in recent anti-CD7 CAR T cell studies. Here we report that monotherapy using base edited (BE) ‘universal’ donor CAR T cells against CD33, CLL-1, or CD7 delivered inhibition of AML in immunodeficient mice when antigen expression was homogenous, but combined use of BE-CAR33 and BE-CARCLL-1 T cells was required to address heterogenous CLL-1 -/+ CD33 -/+ disease. We also demonstrate that removal of shared CD7 antigens enabled compatibility of BE-CAR33 with BE-CAR7 T cells, including in a patient-derived xenograft (PDX) model of AML. Therapeutic strategies envisage ‘pick and mix’ applications of base edited universal CAR T cells in combinations determined by patient-specific antigen profiles. Such approaches also offer the possibility of deep, cell-based, de-bulking and preparative conditioning ahead of allo-SCT followed by donor-derived reconstitution.

Phototropin, a blue-light sensing serine/threonine kinase, plays a pivotal role in regulating diverse photophysiological processes in both plants and algae. In Chlamydomonas reinhardtii , phototropin (CrPhot) localizes to the eyespot and flagella, coordinating key cellular functions such as phototaxis, photosynthesis, gametogenesis, and chlorophyll biosynthesis. While previous research has identified phototropin interactions with signaling proteins such as channelrhodopsins and light-harvesting complex proteins, many aspects of its interaction network and regulatory mechanisms remain unresolved. In this study, we explored novel interacting protein partners of phototropin and their roles in modulating its regulatory functions in Chlamydomonas reinhardtii . Employing a suite of intraflagellar transport (IFT) mutants of C. reinhardtii such as IFT172, IFT52, IFT88, IFT139, kinesin/dynein, CEP290 etc., we elucidate that phototropin localization within the flagella and eyespot is IFT-mediated. Our study highlights interaction of phototropin with other photoreceptors-channelrhodopsins (ChR1 and ChR2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling protein-14-3-3. CRISPR-Cas9 knockouts of phototropin showed reduced ChR1, 14- 3-3 levels and exhibited impaired photomotility. Moreover, two LOV-domain containing histidine kinases, LOV-HK1 and LOV-HK2, were identified in C. reinhardtii . Gene expression of LOV-HK1 and LOV-HK2 were found to be elevated in UV-light in C. reinhardtii and their genes expression was found to be altered in phototropin CRISPR-Cas9 knockouts. This study provides new insights into phototropin signalosome and highlights molecular mechanisms governing its function. The research outcomes advances our understanding of phototropin trafficking and signal modulation in Chlamydomonas reinhardtii , and sets the stage for further exploration into the broader physiological roles of phototropin in cellular responses. Graphical abstract Phototropin, a blue-light receptor in Chlamydomonas reinhardtii , localizes to the flagella and eyespot, mediates phototaxis and photosynthesis. Its trafficking is mediated by intraflagellar transport (IFT) machinery, with mutations in IFT components (kinesin, dynein, IFT172, IFT52, IFT88, IFT139, CEP290) disrupting phototropin localization. Phototropin interacts with other photoreceptors like channelrhodospins (ChR1/2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling proteins (14-3-3), coordinating light-driven responses. These findings underscore the details of phototropin trafficking and phototropin signaling impacting light-induced physiological processes in C. reinhardtii . Highlights • Phototropin localizes in eyepot and flagella in Chlamydomonas reinhardtii . • Intraflagellar transport (IFT) mutants of C. reinhardtii suggest role of different IFT proteins in phototropin trafficking and localization. • Phototropin interacts with other photoreceptors (ChR1 & ChR2, COP6, LOV-HK1 & LOV-HK2) and signaling proteins (14-3-3), contributing to various physiological processes. • CRISPR-Cas9 knockouts of phototropin showed reduced 14-3-3 protein content and photomotility response in C. reinhardtii .

Multiplex genome editing via CRISPR is a powerful tool for the simultaneous knockout, activation, and/or repression of distinct genes or noncoding sequences. However, current toolkits for multiplex editing lack diversity. Repeated use of the same promoter in multiple expression cassettes complicates construct assembly and has long been a concern for genetic stability in the host organism. Additionally, using unnecessarily long promoters may increase the genetic load and introduce uncertainties that impact CRISPR efficiency. To address these challenges, we present a collection of short Polymerase III (Pol III) promoters of diverse origins to support increasingly sophisticated genome editing applications in dicots.

Amongst the evolutionary innovations of many succulents is a photosynthetic lifestyle, where stomatal gas exchange is decoupled from light-dependent carbon fixation. Stomatal complexes in the emerging succulent model Kalanchoë laxiflora consist of two guard cells surrounded by three anisocytic subsidiary cells (SCs). Here, we show that these SCs shuttle ions and thus likely support stomatal movements. Furthermore, gene editing, reporter lines and protein overexpression implicate the stomatal transcription factor MUTE in facilitating additional rounds of asymmetric divisions that form SCs in succulents. This is opposite to the role of MUTE in Arabidopsis thaliana , where it stops rather than induces asymmetric divisions, but reminiscent of MUTE’s SC-related function in grasses. Together, our work deciphers an intricate genetic mechanism that generates innovative stomatal morphology in Crassulaceae succulents.

Latrophilin-3 (LPHN3) is a brain specific adhesion G-protein coupled receptor associated with elevated risk of attention deficit hyperactivity disorder (ADHD). We developed a global Lphn3 knock-out (gKO) rat using CRISPR/Cas9 to delete exon-3. Here we report the development of a floxed Lphn3 rat crossed with tyrosine hydroxylase ( Th-Cre ) rats to create a conditional Lphn3 KO rat specific for catecholaminergic- positive cells. The gKO rats are hyperactive and have egocentric and allocentric navigation deficits but showed sparing of conditioned contextual and novel object recognition memory. Here we compared gKO and cKO rats controlling for litter effects. Both gKO and cKO rats were hyperactive and were impaired in egocentric navigation in the Cincinnati water maze (CWM) with deficits greater in gKO rats. The gKO rats were impaired in allocentric navigation in the Morris water maze (MWM) whereas cKO rats were only slightly affected compared with WT, cre, and floxed rats. Striatal tyrosine hydroxylase and dopamine D1 receptors were not significantly different in either model, nor were NMDA-NR1 or NMDA-NR2 in the hippocampus. We previously showed, however, that dopamine is released more rapidly in the striatum of gKO rats by fast- scan cyclic voltammetry. The cKO model shows an important role of catecholamines in the phenotype of LPHN3 disruption and add evidence that this synaptic protein plays a role in neuroplasticity that are consistent with ADHD.

Abstract Apurinic/apyrimidinic endonuclease 1 (APE1) is a critical enzyme in the base excision repair (BER) pathway, essential for preserving cellular equilibrium. Variations in APE1 activity within blood or tissues can provide significant insights for clinical cancer screening and disease diagnosis. Consequently, the detection of APE1 activity is critical for clinical diagnostics. However, there is currently a deficiency in rapid, straightforward, and sensitive methods for APE1 detection. To address this issue, we developed a method that integrates nicking enzyme assisted amplification (NEAA) with CRISPR-Cas12a signal amplification, enabling one-pot detection of APE1 activity. This method utilizes NEAA to produce a substantial quantity of target DNA that is complementary to the crRNA, thereby triggering the trans-cleavage activity of Cas12a. The activated Cas12a then amplifies and emits signals by cleaving the reporter probe. Our strategy allows for the swift and precise detection of APE1, with a detection threshold of 1×10 − 6 U/mL and a linear detection range of 5×10 − 6 to 0.1 U/mL. It has been effectively utilized for the detection of APE1 in biological samples.

A variety of bacterial anti-phage systems have recently been discovered 1–3 , but how these systems synergize to defend against diverse phages remains poorly understood. Here, we report that the adaptive immune system CRISPR-Cas supervises the expression of diverse immune systems by exploiting the regulatory CRISPR RNA-like RNAs (crlRNAs). The crlRNAs target and inhibit the promoters of various immune systems, including the newly characterized Nezha and Gabija, as well as eight previously unrecognized systems that feature distinct defensive domains. Notably, CRISPR regulation balances the expression level of these systems to ensure effective anti-phage activity while avoiding their autoimmunity risks. In return, the supervised immune systems trigger abortive infections when CRISPR-Cas is inhibited by viral anti-CRISPR proteins, thereby offering an anti-anti-CRISPR protection at the population level. Moreover, these systems complement CRISPR immunity with a differing anti-phage profile. These findings highlight the pivotal role of CRISPR-Cas in orchestrating a diverse range of immune systems and showcase the delicate synergy among the multilayered defense strategies in prokaryotes.

Mammalian genomes contain millions of regulatory elements that control the complex patterns of gene expression. Previously, The ENCODE consortium mapped biochemical signals across many cell types and tissues and integrated these data to develop a Registry of 0.9 million human and 300 thousand mouse candidate cis-Regulatory Elements (cCREs) annotated with potential functions 1 . We have expanded the Registry to include 2.35 million human and 927 thousand mouse cCREs, leveraging new ENCODE datasets and enhanced computational methods. This expanded Registry covers hundreds of unique cell and tissue types, providing a comprehensive understanding of gene regulation. Functional characterization data from assays like STARR-seq, MPRA, CRISPR perturbation, and transgenic mouse assays now cover over 90% of human cCREs, revealing complex regulatory functions. We identified thousands of novel silencer cCREs and demonstrated their dual enhancer/silencer roles in different cellular contexts. Integrating the Registry with other ENCODE annotations facilitates genetic variation interpretation and trait-associated gene identification, exemplified by discovering KLF1 as a novel causal gene for red blood cell traits. This expanded Registry is a valuable resource for studying the regulatory genome and its impact on health and disease.