Biology subjects

Zhao, P.

Publications and source records attributed to Zhao, P..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Identifying crosstalks among post-translational modifications in lung cancer proteomic data

Post-translational modifications (PTMs) are pivotal in cellular regulations, and their crosstalk is related to various diseases such as cancer. Given the prevalence of PTM crosstalk within close amino acid ranges, identifying peptides with multiple PTMs is essential. However, this task is an NP-hard combinatorial problem with exponential complexity, posing significant challenges for existing analysis methods. Here, we introduce PIPI-C (PTM-Invariant Peptide Identification with a Combinatorial model), a novel search engine that addresses this challenge through a mixed-integer linear programming (MILP) model, thereby overcoming the limitations of existing approaches that struggle with high-order PTM combinations. Rigorous validation across diverse datasets confirms PIPI-Cs superior performance in detecting PTM crosstalks. When applied to over 72 million mass spectra of three human cancers--lung squamous cell carcinoma (LSCC), colorectal adenocarcinoma (COAD), and glioblastoma (GBM)--PIPI-C reveals significantly upregulated PTM crosstalks. In LSCC, 50% of 860 upregulated unique PTM site patterns (UPSPs) (when comparing cancer vs. normal samples) carried at least two PTMs, including literature-supported crosstalks such as di-methylation with trifluoroleucine substitution and amidation with proline-to-valine substitution. Similar findings in COAD and GBM highlight PIPI-Cs utility in uncovering cancer-relevant PTM crosstalk landscapes. Overall, PIPI-C provides a robust mathematical framework for decoding complex PTM patterns, advancing our understanding of PTM-driven cellular processes in diseases.

bioinformatics↗

bioRxiv2024

Intra-subgenome regulation induces unbalanced expression and function among bread wheat homoeologs

The differential expression of homoeologous genes confers expression plasticity and facilitates the adaptation and domestication of major polyploid crops. However, how this homoeolog expression bias (HEB) is regulated remains elusive. Here, transcriptome analysis of 406 wheat (Triticum aestivum; AABBDD) accessions revealed great variation in HEB among accessions. We identified 14,727 QTLs regulating HEB (hebQTLs), indicating that HEB is genetically regulated and can be predicted using genotyping data. The hebQTLs only regulate the expression of homoeologs in the same subgenome and downregulate their expression to result in HEB, suggesting that intra-subgenomic rather than inter-subgenomic interactions induce HEB. Furthermore, non-hebQTL-regulated homoeologs have stronger biological functions, are under higher selection pressure and exhibit lower genetic diversity than hebQTL-regulated homoeologs and compensate for the downregulated expressions of hebQTL-regulated homoeologs. Our findings reveal how homoeolog expression is coordinated at the genetic level and provide an avenue for leveraging HEB to improve polyploid crops.

genomics↗

bioRxiv2024

NEK1 modulates neurite outgrowth in motor neurons through coordinating retromer formation

Loss of function of a cell cycle-associated gene NEK1 causes amyotrophic lateral sclerosis (ALS), but how this leads to motor neuron degeneration is unknown. We studied the function of NEK1 in human stem cell-derived motor neurons and found that loss of NEK1 causes decreased neurite length accompanied by transcriptional alterations. We also found that NEK1 interacts with and modulates the formation of the retromer, and that impaired retromer function contributes to neurite outgrowth deficits. We identified SMC3, which interacts with NEK1 during the cell cycle, as a kinase substrate of NEK1 in motor neurons. Notably, loss of SMC3 not only recapitulates the decreased neurite outgrowth, but also affects retromer formation. We suggest that NEK1 interacts with multiple proteins in postmitotic neurons to coordinate retromer formation, and that loss of this leads to impaired neurite outgrowth.

neuroscience↗

bioRxiv2024

The visuomotor transformations underlying defensive behaviors and hunting

The visual system can process diverse stimuli and make the decision to execute appropriate behaviors, but it remains unclear where and how this transformation takes place. We imaged the zebrafish visual system while larvae responded with hunting, freezing, and escape behaviors, and systematically identified visually driven neurons and behaviorally correlated sensorimotor neurons. Our analyses indicate within the optic tectum, broadly tuned sensory neurons are functionally connected to sensorimotor neurons that respond specifically during one behavior, transforming visual information into motor output. We also identified sensorimotor neurons in four other areas downstream of the tectum, and these neurons are also specific for one behavior, indicating that once the decision to behave has been made, the segregation of the pathways continues in later areas. Our findings suggest that the tectum receives visual sensory information and is responsible for selecting a single behavioral outcome, which is then relayed to downstream areas. Significance statementHere, we developed a novel visually-evoked freezing paradigm in zebrafish, and combined this with escape and hunting behaviors to ask how visual stimuli are identified and converted into different behavioral outcomes. We found that the optic tectum contains neurons that detect all three stimuli, as well as sensorimotor neurons for the three behaviors, suggesting that it is a site of sensorimotor transformation, which was supported by our analysis of correlations between the populations. The sensorimotor neurons in the tectum are highly specific for one behavior, and this segregation is maintained in the three downstream areas where we also identified sensorimotor neurons, indicating that the tectum flexibly transforms visual information into a single behavioral output.

neuroscience↗

bioRxiv2024

rarestR: An R package using rarefaction metrics to estimate α-diversity (species richness) and β-diversity (species shared) for incomplete samples

Species abundance data is commonly used to study biodiversity patterns. In this context, estimating - and {beta}-diversity based on incomplete samples can lead to undersampling biases. It is therefore essential to employ methods that enable accurate comparisons of - and {beta}-diversity across varying sample sizes. This involves relying on biodiversity measures that are focused on accurately estimating the total number of species within a community, as well as the total number of species shared by two communities. Rarefaction offers such a method, where -diversity is estimated for standardized sample sizes. Rarefaction methods can also be used as a basis for {beta}-diversity calculations for standardized sample sizes. In this application note, we introduce a new R package, rarestR, designed to estimate abundance-based - and {beta}-diversity measures for inconsistent samples using rarefaction metrics. Additionally, the package offers parametric extrapolations to estimate the total expected number of species within a single community and the total expected number of species shared between two communities. Furthermore, it provides visualization for the curve fitting associated with these estimators. Overall, the rarestR package is useful in estimating - and {beta}-diversity values for incomplete samples, for example in studies involving highly mobile or species-rich taxa. These species estimators offer a complementary approach to non-parametric methods, such as the Chao series of estimators.

ecology↗

bioRxiv2024

Searching Post-translational Modifications in Cross-linking Mass Spectrometry Data

Cross-linking mass spectrometry (XL-MS) is a technique for investigating protein-protein interactions (PPIs) and protein structures. In the realm of biology, post-translational modifications (PTMs) play a critical role in regulating PPIs and reshaping protein structures. However, the identification of PTMs in XL-MS data poses a great computational challenge and thus remains unexplored. In this study, we introduce SeaPIC, the first XL-MS tool that enables biologists to investigate PTMs in PPIs and protein structures. Our experiments demonstrate the successful identification of PTMs within cross-linked peptides, which were previously undiscovered.

bioinformatics↗

bioRxiv2024

Increased dosage of wild-type KRAS protein drives KRAS-mutant lung tumorigenesis and drug resistance

Almost 30% of lung adenocarcinomas are driven by activating KRAS mutations. The heterogeneous clinical behavior observed in these cancers could be due to the imbalance of wild-type and oncogenic KRAS alleles. However, the role of RAS dysregulation at the protein level needs to be further explored. A genome-wide global protein stability screen identified the CUL3 ubiquitin ligase adaptor LZTR1, as a major proteostatic regulator of wild-type but not mutant KRAS. In KRAS-mutant lung adenocarcinoma, shallow deletion of LZTR1 is observed in up to 50% of patients and is associated with hypoxic signatures and poorer progression-free disease survival. In a Kras-mutant lung cancer mouse model, heterozygous loss of Lztr1 promoted tumor growth, led to peritumoral vascular remodeling, and limited the response to the KRAS-G12D inhibitor MRTX1133. The vascular alteration in LZTR1-depleted lung cancer was mediated by increased wild-type KRAS protein dosage, which promoted mTOR pathway activation and a subsequent increase in VEGFA secretion. The inhibition of the PI3K/mTOR pathway using dactolisib normalized tumor vasculature, improved drug delivery, and overcame resistance to KRAS-G12D inhibitors. In summary, the dysregulation of RAS proteostasis contributes to lung tumorigenesis, and targeting wild-type KRAS signaling is crucial to overcome intrinsic resistance to inhibitors of mutant KRAS. SignificanceOur study highlights the impact of RAS proteostasis on lung cancer development and progression. Vascular normalization achieved by suppressing wild-type KRAS signaling improved the delivery and response to MRTX1133 in KRAS-mutant lung cancer with shallow LZTR1 deletion.

cancer biology↗

bioRxiv2024

Exploring the repository of de novo designed bifunctional antimicrobial peptides through deep learning

Antimicrobial peptides (AMPs) are attractive candidates to combat antibiotic resistance for their capability to target bio-membranes and restrict a wide range of pathogens. It is a daunting challenge to discover novel AMPs due to their sparse distributions in a vast peptide universe, especially for peptides that demonstrate potencies for both bacterial membranes and viral envelopes. Here we establish a de novo AMP design framework by bridging a deep generative module and a graph-encoding activity regressor. The generative module learns hidden grammars of AMP features and produces candidates sequentially pass antimicrobial predictor and antiviral classifiers. We discover 16 bifunctional AMPs and experimentally validated their abilities to inhibit a spectrum of pathogens in vitro and in animal models. Notably, P076 is a highly potent bactericide with the minimal inhibitory concentration of 0.21 M against multidrug-resistant A. baumannii, while P002 broadly inhibits five enveloped viruses. Our study provides feasible means to uncover sequences that simultaneously encode antimicrobial and antiviral activities, thus bolstering the function spectra of AMPs to combat a wide range of drug-resistant infections.

microbiology↗

bioRxiv2024

Arginine Di-methylation of RIPK3 Safeguards Necroptosis for Intestinal Homeostasis

The necroptosis mediated by RIPK3 is stringently regulated for intestinal homeostasis. Here we found that mice lacking Prmt5 (Protein arginase methyltransferase 5) in intestinal epithelial cells (IECs) caused premature death with IECs necroptosis, villus atrophy and loss of Paneth cells. This pathology can be partially rescued by antibiotic treatment, germ-free breeding condition and pharmaceutical inhibition of RIPK1 and RIPK3, but aggravated for embryonic lethality by Caspase-8 deficiency, which demonstrating the importance of commensal bacteria and necroptosis for the Prmt5-IEC deficiency. Intriguingly, tumor-necrosis factor (TNF) receptor 1(Tnfr1) deficiency could not completely rescue the pathology, and mice deficit in Z- DNA binding protein 1(ZBP1) exhibited shorter lifespan compared with Prmt5 null mice, suggesting Prmt5 loss might trigger TNFR-RIPK1-depenfent and ZBP1- dependent necroptosis. Mechanically, we identified the 479-arginine residue of RIPK3 di-methylated by PRMT5 was an endogenous checkpoint for necroptosis. Furthermore, RIPK3-R479K mutation had higher affinity with both RIPK1 and ZBP1 by immunoprecipitation and STORM (Stochastic Optical Reconstruction Microscopy) analysis, which might explain the endogenous necroptosis triggered by mutated RIPK3 even without upstream stimuli. Moreover, the peptide of RIPK3-SDMA (Symmetric dimethylarginine of 479) could rescue lethality of Prmt5 lacking mice through necrosome formation inhibition, which demonstrating the great potential for necroptosis-related disease treatment through RIPK3 dimethylation targeting.

cell biology↗

bioRxiv2024

Fast and accurate modeling and design of antibody-antigen complex using tFold

Accurate prediction of antibody-antigen complex structures holds significant potential for advancing biomedical research and the design of therapeutic antibodies. Currently, structure prediction for protein monomers has achieved considerable success, and promising progress has been made in extending this achievement to the prediction of protein complexes. However, despite these advancements, fast and accurate prediction of antibody-antigen complex structures remains a challenging and unresolved issue. Existing end-to-end prediction methods, which rely on homology and templates, exhibit sub-optimal accuracy due to the absence of co-evolutionary constraints. Meanwhile, conventional docking-based methods face difficulties in identifying the contact interface between the antigen and antibody and require known structures of individual components as inputs. In this study, we present a fully end-to-end approach for three-dimensional (3D) atomic-level structure predictions of antibodies and antibody-antigen complexes, referred to as tFold-Ab and tFold-Ag, respectively. tFold leverages a large protein language model to extract both intra-chain and inter-chain residue-residue contact information, as well as evolutionary relationships, avoiding the time-consuming multiple sequence alignment (MSA) search. Combined with specially designed modules such as the AI-driven flexible docking module, it achieves superior performance and significantly enhanced speed in predicting both antibody (1.6% RMSD reduction in the CDR-H3 region, thousand times faster) and antibody-antigen complex structures (37% increase in DockQ score, over 10 times faster), compared to AlphaFold-Multimer. Given the performance and speed advantages, we further extend the capability of tFold for structure-based virtual screening of binding antibodies, as well as de novo co-design of both structure and sequence for therapeutic antibodies. The experiment results demonstrate the potential of tFold as a high-throughput tool to enhance processes involved in these tasks. To facilitate public access, we release code and offer a web service for antibody and antigen-antibody complex structure prediction, which is available at https://drug.ai.tencent.com/en.

bioinformatics↗

bioRxiv2024

Unraveling wheat endosperm development: epigenetic regulation and novel regulators for enhanced yield and quality

Starch content and seed storage protein (SSP) composition are critical factors influencing wheat grain yield and quality. To uncover the molecular mechanisms governing their biosynthesis, we conducted transcriptome and epigenome profiling across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlighted the crucial role of accessible promoter regions as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. By integrating time-serial RNA-seq and ATAC-seq data, we constructed a hierarchical transcriptional regulatory network (TRN) governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibited overlap with genetic regions associated with grain size and quality traits, and their functional significance was validated through expression-phenotype association analysis among wheat accessions and TILLING mutants. In-depth functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrated its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. An elite haplotype of TaABI3-A1 with higher grain weight was identified during the breeding process in China, and its superior trait was associated with altered TaABI3-A1 expression levels. Additionally, we identified the potential upstream regulator, wheat GAGA-binding transcription factor 1 (TaGBP1), influencing TaABI3-A1 expression. Our study provides novel and high-confidence regulators, presenting an effective strategy for understanding the regulation of SSP and starch biosynthesis and contributing to breeding enhancement.

plant biology↗

bioRxiv2023

TaNF-Y-PRC2 orchestrates temporal control of starch and protein synthesis in wheat

The endosperm in cereal grains is instrumental in determining grain yield and seed quality, as it controls the production of starch and protein. In this study, we identified a specific TaNF-Y trimeric complex, consisting of TaNF-YA3-D, TaNF-YB7-B, and TaNF-YC6-B, exhibiting robust expression within endosperm during grain filling stage in wheat. Knock-down of either TaNF-YA3 or TaNF-YC6 led to less starch but more gluten proteins. Detailed analyses have unveiled that the TaNF-Y indirectly boosts starch biosynthesis genes by reducing TaNAC019, a repressor of TaAGPS1a, TaSuS2, thereby regulating starch biosynthesis. Conversely, the TaNF-Y directly inhibits the expression of gliadin and low molecular weight (LMW)-GS coding genes, including TaGli-{gamma}-700 and TaLMW-400. Furthermore, the TaNF-Y components interact with TaSWN, the histone methyltransferase subunit of Polycomb repressive complex 2 (PRC2), to repress the expression of TaNAC019, TaGli-{gamma}-700, and TaLMW-400 through H3K27me3 modification. Notably, weak mutation of TaFIE, core subunit of PRC2, has reduced starch but elevated gliadin and LMW-GS levels. Intriguingly, DNA variations of TaNF-Y components are widely associated with seed developmental traits. In particular, variation within the coding region of TaNF-YB7-B is linked to differences in starch and protein content. Distinct haplotypes of TaNF-YB7-B affect its interaction with TaSWN, influencing the repression of targets like TaNAC019 and TaGli-{gamma}-700. Our findings illuminate the intricate molecular mechanisms governing epigenetic regulation by the TaNF-Y-PRC2 for wheat endosperm development. Manipulating the TaNF-Y complex holds potential for optimizing grain yield and enhancing quality.

plant biology↗

bioRxiv2023

Biosynthesis of the proteins containing neurotoxin β-N-methylamino-L-alanine in marine diatoms

Neurotoxin {beta}-N-methylamino-L-alanine (BMAA) has been deemed a pathogenic factor for human neurodegenerative diseases. It is an important issue to disclose the biosynthesis mechanism of BMAA in marine diatoms. In the present study, the iron (Fe) limitation (1/3 x Fe) was found to suppress the growth of diatoms but stimulate the production of BMAA-containing proteins, maximum 7.7 fold in Thalassiosira minima. Transcriptome analysis showed that energy metabolism, protein biosynthesis and carbon fixation functions were mainly affected by the Fe limitation in the diatom. Analysis of subcellular distribution of BMAA showed that BMAA-containing proteins were mainly detected in the endoplasmic reticulum and the Golgi apparatus. Combination results of the responses of the diatom to Fe deficiency and co-culture with cyanobacteria in our previous study, we speculate that cysteine embedded in peptide chains and methylamine produced by the diatom itself are possibly catalyzed by the cysteine synthase (cysK) to form the BMAA structure in situ. Spiked methylamine in culture media significantly stimulated the production of BMAA, and BMAA amounts were correlated with the expression of cysK gene in different diatoms. The reduced ubiquitination-mediated proteolysis and vesicle trafficking precision through the COPII system would aggravate the accumulation of BMAA-containing proteins in the diatom. Significance StatementWith the detection of neurotoxin BMAA in diverse marine diatoms, the pathogenic risk of BMAA has been further concerned to human neurodegenerative diseases such as Alzheimers disease. Interestingly, BMAA-containing proteins are the dominant forms of this neurotoxin in diatoms. It is a keystone issue to disclose the biosynthesis mechanism of BMAA in marine diatoms. We found Fe-limitation could stimulate the production of BMAA-containing proteins in diatoms and explored its biosynthesis using transcriptomics in this study. Results suggested that cysteine embedded in peptides and methylamine in cytoplasm were catalyzed by the cysteine synthase (cysK) to form BMAA. This study hints that the biosynthesis of BMAA would be improved by the worldwide prevalence of iron deficiency in the coastal waters.

biochemistry↗

bioRxiv2023

Millimeter-scale magnetic implants paired with a fully integrated wearable device for wireless biophysical and biochemical sensing

Implantable sensors can directly interface with various organs for precise evaluation of health status. However, extracting signals from such sensors must rely on transcutaneous wires, integrated circuit chips, or cumbersome readout equipment, which increases the risks of infection, reduces the biocompatibility, or limits the portability. Here, we develop a set of millimeter-scale, chip-less and battery-less magnetic implants that can measure biophysical and biochemical signals wirelessly. In particular, the implants form two-way communications with a fully integrated wearable device, where the wearable device can induce a large-amplitude damped vibration of the magnetic implants and capture their subsequent motions in a wireless manner. Such damped vibrations reflect not only the biophysical conditions surrounding the implants movements, but also the concentration of a specific biochemical depending on the surface modification. Experiments in rat models demonstrate the capabilities in measuring cerebrospinal fluid (CSF) viscosity, intracranial pressure (ICP), and CSF glucose levels. This miniaturized system opens possibility for continuous, wireless monitoring of a wide range of biophysical and biochemical conditions within the living organism.

bioengineering↗

bioRxiv2023

Predicting protein folding pathway using a novel folding force field model derived from known protein universe

The protein folding problem has emerged as a new challenge with the significant advances in deep learning driven protein structure prediction methods. While the structures of almost all known proteins have been successfully predicted, the process by which they fold remains an enigma. Understanding the intricate folding mechanism is of paramount importance, as it directly impacts the stable expression and biological function of proteins. Here, we propose FoldPAthreader, a protein folding pathway prediction method that designs a novel folding force field model by exploring the intrinsic relationship between protein evolutionary history and folding mechanisms from the known protein universe. Further, the folding force field is used to guide Monte Carlo conformational sampling, driving the protein chain fold into its native state by exploring a series of transition states and potential intermediates. On the 30 targets we collected, FoldPAthreader can successfully predict 70% of the proteins whose folding pathway is consistent with wet-lab experimental data. The results show that the folding force field can capture key dynamic features of hydrogen bonding and hydrophobic interactions. Importantly, for the widely studied BPTI and TIM proteins, the folding pathway predicted by FoldPAthreader have the same microscopic dynamic properties as those simulated by molecular dynamics. Significance StatementProtein folding is the process by which a protein acquires its functional conformations by gradually transforming from random coils into a specific three-dimensional structure. In the post-Alphafold2 era, functional analysis of protein macromolecules should not only rely on the final state structure, but should pay more attention to the structural folding process, that is, the various intermediate states formed during the folding process. At present, there is no folding force field specifically used for protein folding pathway prediction in computational biology. Here we extracted folding information from 100-million-level structure database and designed a new folding force field for folding pathway prediction, proving a hypothesis that the protein evolutionary history implicitly contains folding information of individual protein. This study may provide new insights into the understanding of protein folding mechanisms, which is expected to advance drug discovery.

bioinformatics↗

bioRxiv2023

TIPE drives a cancer stem-like phenotype by promoting glycolysis via PKM2/HIF-1alpha axis in melanoma

TIPE (TNFAIP8) has been identified as an oncogene and participates in tumor biology. However, how its role in the metabolism of tumor cells during melanoma development remains unclear. Here, we demonstrated that TIPE promoted glycolysis by interacting with pyruvate kinase M2 (PKM2) in melanoma. We found that TIPE induced PKM2 dimerization, thereby facilitating its translocation from the cytoplasm to the nucleus. TIPE-mediated PKM2 dimerization consequently promoted HIF-1 activation and glycolysis, which contributed to melanoma progression and increased its stemness features. Notably, TIPE specifically phosphorylated PKM2 at Ser 37 in an ERK-dependent manner. Consistently, the expression of TIPE was positively correlated with the levels of PKM2 Ser37 phosphorylation and cancer stem cell markers in melanoma tissues from clinical samples and tumor bearing mice. In summary, our findings indicate that the TIPE/PKM2/HIF-1 signaling pathway plays a pivotal role in promoting cancer stem cell properties by facilitating the glycolysis, which would provide a promising therapeutic target for melanoma intervention.

cancer biology↗

bioRxiv2023

Modern wheat breeding selection synergistically improves above- and below-ground traits

The root system, as a fundamental organ for uptaking water and nutrients and interacting with the local environmental conditions, has been postulated to be the foundation for a second Green Revolution. However, the status of the root system during modern wheat breeding remains to be elucidated. Here, by analyzing the phenotypes of 406 wheat accessions on a large scale, we found the root systems of modern cultivars were synchronisely changed along with the above-ground traits. Furthermore, the genomic blocks with phenotypic effects on both above- and below-ground traits were observed to be enriched in the selection sweeps, highlighting that modern wheat breeding contributed to the synchronised changes. More importantly, the haplotypes selected by breeders within the selection sweeps synergistically improved both the above- and below-ground traits, suggesting that modern wheat breeding has improved the root system indirectly, which may contribute to the higher grain yields of modern wheat cultivars. Our results demonstrated that modern wheat breeding synergistically improved the above- and below-ground traits.

genetics↗

bioRxiv2023

Rationale design of unrestricted pRN1 derivatives and their application in construction of a dual plasmid vector system for Saccharolobus islandicus

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, including efficient genome editing, gene silencing and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are solely based on the pRN2 cryptic plasmid. Although this plasmid co-exists with pRN1 in their original host, early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa. islandicus. Herein we identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (TargetN1). Mutated targets were then designed (N1a, N1b, N1c) and tested for their capability of escaping from the host CRISPR immunity by using plasmid interference assay. This revealed that the original target triggers the CRISPR immunity in this archaeon whereas all three mutated targets do not, indicating that all designed target mutations evade the host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability of replication. In addition, pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model. Impact statementWhen pRN1 was employed for vector construction in Saccharolobus islandicus REY15A, pRN1-derived vectors are not stable in this archaeon. Here we show that pRN1 orf904 encoding a putative replicase on pRN1 carries a DNA segment to be targeted by the host I-A CRISPR system. By designing mutated target sequences that evade the CRISPR immunity, efficient plasmid vectors were obtained with mutated pRN1 backbones. This strategy could be applied in developing host-vector systems for other microorganisms with plasmids or viruses carrying CRISPR target sequences. Moreover, the resulting dual vector system would facilitate genetic studies with this crenarchaeal model.

microbiology↗