348 results about "Recombinase" patented technology

The invention discloses a kit for detecting tomato brown crinkled fruit virus based on enzyme-mediated dual-amplification nucleic acid amplification and a detection method. The detection system comprises a primer pair DNA (deoxyribonucleic acid) upstream primer (F5) and a DNA downstream primer (R6) for amplifying the tomato brown crinkled fruit virus movement protein gene, a probe (RNA primer (RNA1)) for detecting the MP gene, a nucleic acid amplification group enzyme system (recombinase, single-chain binding protein, polymerase and ATP (adenosine triphosphate) energy regeneration enzyme), a signal amplification group enzyme system (positioning enzyme and cutting enzyme), activating enzyme NTP, a buffer solution and RNase-free Water. The system composed of the freeze-drying protective agent is prepared into the pre-subpackaged freeze-dried balls. The kit disclosed by the invention can realize DNA detection of aM concentration, and besides rapid qualitative detection of the tomato brown crinkled fruit virus, early screening of the tomato brown crinkled fruit virus can also be realized due to high detection sensitivity.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

The invention belongs to the technical field of biology, and particularly relates to an SLC44A1 gene and / or SLC44A5 gene knockout animal model as well as a construction method and application thereof. The construction method of the animal model comprises the following steps: deleting exon regions from the fourth exon region to the thirteenth exon region of the SLC44A1 gene and / or the second exon region of the SLC44A5 gene in mouse brain oligodendroglia cells in a high-efficiency manner by utilizing gene editing and a Cre-loxP recombinase system, so as to realize the knockout of a target gene. The animal model constructed by the invention can be used for researching the regulation mechanism of the choline transporters (SLC44A1 and SLC44A5) on myelin sheath formation, deeply knowing the biological functions of the choline transporters (SLC44A1 and SLC44A5), simulating the occurrence and development processes of human related diseases, revealing the pathogenesis of the related diseases, providing a new thought for prevention and treatment of brain myelin sheath dysplasia, and providing a new foundation for the prevention and treatment of brain myelin sheath dysplasia. And an effective drug or a treatment mode aiming at the disease is screened by utilizing a gene knockout animal model, so that a new choice is provided for clinical treatment of the disease.

The invention discloses a CRISPR / Cas12a-RPA (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 12a-recombinase polymerase amplification)-based sequence combination for rapidly detecting mouse hepatitis virus and a kit thereof, and belongs to the technical field of biological detection The sequence combination comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence; the kit comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence, the kit further comprises a mouse hepatitis virus negative control standard substance, RNase-free water, an RPA reaction buffer solution, an NEB buffer solution, Lba Cas12a nuclease, dry powder RPA reaction microspheres containing recombinase polymerase and magnesium acetate with the concentration of 280 mM. The kit can be used for rapidly detecting the mouse hepatitis virus. The primer sequence combination provided by the invention is good in conservative property and specificity and relatively high in sensitivity, and rapid detection of the mouse hepatitis virus can be realized.

The invention discloses a method for culturing CIK (Cytokine-Induced Killer) cells and application thereof. The method comprises the following steps: recovering cord blood, mixing and centrifuging the recovered cord blood and a buffer solution, re-suspending and precipitating, centrifuging again to obtain cord blood mononuclear cells, inoculating the cord blood mononuclear cells into an activation culture medium, and culturing to obtain the CIK cells, the buffer solution contains L-proline, human recombinant DNA enzyme and angelica polysaccharide. According to the method, the mononuclear cells can be separated from the umbilical cord blood preserved by liquid nitrogen, the utilization rate of the umbilical cord blood which can only be discarded due to insufficient cells is increased, the traditional lymphocyte separation process is optimized, the recovery rate of the mononuclear cells is increased, and the cost is reduced. The method provided by the invention can be used for culturing dozens to ten billions of CIK cells from a small amount of cryopreserved cord blood, and has huge advantages in the field of CIK cell culture and application.

The invention discloses a construction method and application of a mouse model for specifically tracing heart valve cells, and belongs to the technical field of animal model construction. The model construction method comprises the following steps: (1) constructing a Wid1-Cre hybrid mouse before inserting a Cre recombinase coding gene into a termination codon of a mouse Wid1 gene; and (2) hybridizing the Wid1-Cre hybrid mouse with the Rosa26-tdTomato homozygous report mouse, so as to obtain the double transgenic mouse which simultaneously carries the Wid1-Cre gene and the Rosa26-tdTomato gene. According to the invention, on the basis of the expression characteristic that Wif1 is only limited to a valve area in the heart, Wif1-Cre is constructed; according to the Rosa26-tdTomato mouse model, specific tracing of heart valve cells is achieved, and an important experimental tool is provided for mechanism research of heart valve diseases and development of therapeutic drugs.

The invention discloses a panda-derived feline panleucopenia virus RAA-CRISPRCas12a detection method, and belongs to the technical field of virus detection, and the panda-derived feline panleucopenia virus RAA-CRISPRCas12a detection method comprises the following steps: S1, primer and probe design; s2, screening an RAA amplification primer; s3, RAA reaction condition optimization; s4, performing RAA sensitivity and specificity verification; s5, preparing crRNA (Complementary Ribonucleic Acid); s6, optimal crRNA screening is carried out; s7, optimizing the RAA-CRISPR / Cas12a (Reverse Amplified Architecture- S8, the specificity and the sensitivity of the RAA-CRISPR / Cas12a are verified; s9, clinical sample detection; according to the invention, by elaborately designing a primer and a probe aiming at the capsid protein VP2 gene of the feline parvovirus (FPV), fully utilizing the advantages of recombinase-mediated amplification (RAA) and a CRISPR / Cas12a system, and comprehensively optimizing reaction conditions and components of the system, the detection method has remarkable advantages, can quickly complete the detection process under the condition of constant temperature of 37 DEG C, is extremely high in sensitivity, and can be used for rapidly detecting the feline parvovirus. The lowest detection limit can reach 1 * 10 < 1 > copies / mu L, the specificity is high, and the coincidence rate with a real-time fluorescent quantitative PCR method is as high as 100%.

The invention relates to the technical field of biology, and particularly discloses a method for detecting corn leukoderma pathogenic bacteria based on RPA-LFD (Recombinase Polymerase Amplification-Lateral Flow Dipstick), which comprises the following steps: a recombinase polymerase amplification RPA reaction system comprising an upstream primer PA-F2, a biotin-labeled downstream primer PA-R3-biotin and an nfo probe PA3-nfo-2; the lateral flow chromatography analysis LFD test strip is used for detecting an amplification product; a reaction buffer solution and a dilution buffer solution; a rapid and visual corn leaf spot detection method is developed by using a recombinase-mediated isothermal amplification (PRA) technology and combining an nfo probe with a lateral flow chromatography analysis and diagnosis method, and finally, an efficient and rapid corn leaf spot detection, prevention and control system is established by popularizing and applying a novel visual detection method in a field.

The invention provides a primer group and a kit for detecting bovine mycobacterium paratuberculosis based on a recombinase isothermal amplification technology and application, and relates to the technical field of biological detection.The primer group comprises a forward primer MAP-F3, a reverse primer MAP-R3 and a specific fluorescent probe MAP-P3 and has good specificity; the sensitivity reaches 10fg / mu L, which is more sensitive than that of a colloidal gold test strip, the dependence on equipment is broken through, and the detection result is more accurate. The technical problem that in the prior art, it is difficult to distinguish bovine mycobacterium paratuberculosis from other pathogens through a detection method, or the detection process is tedious is solved. According to the prepared reagent or kit, the liquid preparation process is simple, the whole amplification process can be completed only in 30 min, the amplification yield can reach 109-1010 copies, the method is rapid and efficient, the amplification result can be directly judged according to fluorescence data, other auxiliary means are not needed, and the method is suitable for field detection.

The invention discloses a system for controllably inducing pyroptosis of tumor cells and application of the system. The system comprises a red light activated Cre recombinase system and an expression vector of a GSDMDNT gene sequence of loxP flanking; the Cre recombinase system comprises: (1) an expression vector 1 containing a photosensitive protein PhyA gene sequence and a CreN59 sequence, and a connecting peptide located between the PhyA gene sequence and the CreN59 sequence; (2) an expression vector 2 containing a nucleoplasm shuttle protein FHY1 gene sequence and a CreC60 sequence, and a connecting peptide located between the FHY1 gene sequence and the CreC60 sequence; and (3) phycocyanobilin is contained. The system disclosed by the invention can be used for artificially and controllably regulating and controlling the expression of the cytotoxic gene GSDMDNT, so that directional and controllable pyroptosis induction is realized. The method can be applied to the fields of tumor therapy, gene therapy, cell biology and the like, and particularly has a wide application prospect in application scenarios requiring accurate control of gene expression.

The invention belongs to the technical field of biological medicine, and particularly relates to an engineered cell microvesicle and a delivery system based on the engineered cell microvesicle, the system realizes efficient preparation of 1-5 [mu] m cell microvesicles, and the cell microvesicles have a large space volume and can be used for preparing the cell microvesicles. The carrier can be used for loading and delivery of target protein, polypeptide and recombinase which are specifically expressed in mother cells. The system transfects mother cells through lentivirus transfection or plasmid transfection to further produce cell microvesicles, and the microvesicles can load more goods and inherit membrane proteins of the mother cells, and can also effectively load intracellular proteins to realize effective delivery. By virtue of good structural stability, high immunogenicity and excellent biocompatibility, the cell microvesicle reduces systematic toxic and side effects of a traditional carrier, is expected to become an effective drug delivery system, and has great application potential in the field of gene therapy.

The invention discloses a method for site-specific replacement or insertion of DNA fragments in a plant genome, and belongs to the technical field of biology. The method disclosed by the invention comprises the following steps: editing a genome of a plant protoplast by adopting a CRISPR / Cas9 system and taking a double-stranded DNA repair template as a donor template to obtain an edited protoplast; and carrying out culture regeneration on the edited protoplast by utilizing a plant protoplast transformation method to obtain a site-directed mutagenesis plant in the genome so as to realize site-directed mutagenesis of the target plant genome. The method disclosed by the invention can be used for accurately replacing DNA in a plant genome at a fixed point. According to the invention, a recognition sequence of recombinase can be accurately inserted into a genome at a fixed point through homologous recombination, and then accurate fixed-point replacement of a gene large fragment is realized by virtue of a specific recombinase system.

The invention discloses a set of LSR gene recombination system for plants, and belongs to the technical field of biology. The recombinant system comprises Kp03 macroserine recombinase and attP and attB corresponding to the Kp03 macroserine recombinase, and efficient gene knock-in editing can be realized in rice and arabidopsis thaliana cells without additional cofactors. The gene editing protein Kp03 can show efficient plasmid level gene knock-in capability in rice and arabidopsis thaliana cells, a 27kb large DNA fragment can be inserted into an attB target site to the maximum extent, and effective gene knock-in editing can still be performed after an attB sequence is truncated to 15bp, so that the application range of gene editing is expanded. In addition, 3.4 kb exogenous DNA fragments can be successfully inserted into the rice genome, a new gene editor is provided for plant large fragment gene knock-in, and the gene knock-in method has important application value.

SpRYc is a grafted ScCas9++-SpRY chimeric Cas9 possessing minimal 5′-NNN-3′ PAM specificity. SpRYc comprises the N-terminus (residues 1-1119) of ScCas9++ (Sc++), including the flexible loop, followed by the region of SpRY (residues 1111-1368) spanning its PAM-interacting domain mutations. Methods of altering gene expression include use of SpRYc in complex with guide RNA in a CRISPR-Cas9 system.

The invention relates to a nucleic acid detection platform based on an SHERLOCK technology, which is used for diagnosing CAV-2. The SHERLOCK consisting of recombinase polymerase nucleic acid pre-amplification (RPA) and CRISPR-Cas13a is mainly used for specifically recognizing a conserved sequence of CAV-2RNA (Ribonucleic Acid) through crRNA (Complementary Ribonucleic Acid) so as to activate the non-specific'side cutting activity 'of lwCas13a, so that the report RNA is degraded. A real-time fluorescent quantitative PCR (qPCR) system or a lateral flow test strip is used for collecting fluorescent signals generated by cracking of the reporter molecules, and a visual result is displayed. In addition, through HUDSON treatment, RPA-CRISPR can be amplified at room temperature without viral nucleic acid extraction. The detection limit of the method is 1.1 * 10 < 2 > copies / [mu] L, and the method has no cross reaction with other canine infectious viruses. From sample collection to result output, the whole reaction of RPA-CRISPR can be completed within two hours, and the RPA-CRISPR kit is suitable for rapid test of clinical samples and can work well in the field or in primary laboratories. The method can get rid of the limitation of a detection instrument and complex operation, and is suitable for on-site rapid batch detection. The method is preliminarily verified and further optimized and applied.

The invention discloses a preparation method of recombinant catechol-2, 3-dioxygenase and an application of the recombinant catechol-2, 3-dioxygenase. According to the invention, a novel catechol-2, 3-dioxygenase group C23O927 is identified from a constructed metagenome library, and the catechol-2, 3-dioxygenase group C23O927 is a key enzyme for catalyzing degradation of polycyclic aromatic hydrocarbon pollutants. A C23O927-pET32a (+) recombinant expression vector is constructed and converted into host bacteria, heterologous expression of target protein is achieved, and the prepared recombinase belongs to acid enzymes and has good resistance to high temperature, acid, alkali, high-concentration metal ions, organic solvents and surfactants. The recombinase provided by the invention can be used in an acidic environment, has very good enzymatic activity, can catalytically degrade various catechol compounds, can be better used for treating polycyclic aromatic hydrocarbon polluted environments, and has important significance for developing more products which can tolerate various extreme environments and efficiently catalyze degradation of polycyclic aromatic hydrocarbons.

The invention relates to the technical field of technical gene detection, in particular to a recombinase and recognition site screening method and system based on structure classification prediction.The method comprises the steps that to-be-matched recombinase is obtained for structure resolution prediction.The functional domain of the to-be-matched recombinase is obtained; searching in a first database according to the functional domain, searching candidate recombinase of which the similarity meets a preset condition, and screening to obtain optimal recombinase; determining an interaction region of the current large serine recombinase and the target DNA sequence from a second database, and sequentially obtaining interaction amino acid sequences corresponding to the preferable recombinase; and matching with the amino acid characteristic sequence of the current large serine recombinase in the second database to obtain the target large serine recombinase with the highest similarity in the preferred recombinase and the corresponding recognition site sequence. According to the method, the structure of the large protein is subjected to structure comparison mining and splitting, and recognition site prediction is performed on interaction region amino acid characteristics, so that the research and development period is shortened, and the research and development cost is saved.

The invention relates to the field of molecular diagnosis and nucleic acid amplification, in particular to a recombinase polymerization amplification method based on Twinkle helicase enhancement and application of the recombinase polymerization amplification method. According to the invention, Twinkle helicase is introduced into an RPA system for the first time, and primer combination and template unlinking are assisted by utilizing ATP-dependent unlinking activity and DNA annealing activity of the Twinkle helicase, so that the amplification efficiency and the detection sensitivity are remarkably improved. The method is suitable for plasmid DNA, a high GC template and a clinical nucleic acid sample, and can be used for constructing an RPA kit containing Twinkle, so that the application of the RPA kit in pathogen detection, molecular diagnosis and on-site rapid detection is expanded. According to the invention, the defects of the existing RPA technology in sensitivity and stability are overcome, and a new solution is provided for developing a novel nucleic acid self-detection and field detection tool.

The present disclosure relates to enzymes, compositions and methods for performing conditional homologous recombination of a targeted DNA molecule or genome by using modified proteins comprising nucleic acid DNA binding proteins with protease-conditional recombinase (hereinafter “ProRec”) activity. Namely a new cre recombinase pro enzyme, compositions / kit and sensors comprising the cre recombinase pro enzyme of the present disclosure. Namely, a cre recombinase pro-enzyme, for identifying and quantifying proteolytic activity in a disease or an infection, comprising intein mediated circularization, a flip-excision cassette comprising an output reporter and / or effector protein; a linker sequence cleavable by a protease.

The invention belongs to the technical field of biological engineering, and provides curcumin / dihydrocurcumin reductase MaCurA, a coding gene and application. The reductase can efficiently catalyze 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone to synthesize tetrahydrocurcumin, an efficient solution is provided for industrial preparation of tetrahydrocurcumin, and experiments prove that the recombinase can catalyze the synthesis of tetrahydrocurcumin in the catalysis of 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone. In a reaction system for preparing tetrahydrocurcumin from 2, 5-diketone, actually measured enzyme activity is stabilized at 3.38 U / mg level, and compared with a traditional chemical synthesis method, the production efficiency is remarkably improved.

The invention discloses a method for detecting high sensitivity of monkey pox virus by a one-pot method RPA-CRISPR based on temperature control, and the method comprises the following steps: establishing a temperature-controlled RPA and CRISPR-Cas12b combined detection method, which is called as CRATS for short, by utilizing the reaction temperature difference between RPA amplification and CRISPR-Cas12b cutting; the reaction process in a tube system is regulated and controlled by stages through a temperature switching strategy: target amplification is completed at 37 DEG C firstly, then the temperature is raised to 60 DEG C, Cas12b cutting is started, so that sequential separation of amplification and cutting reactions is realized, and finally detection is realized through a fluorescence signal released by molecular beacon breakage. The problem that Cas enzyme cutting activity interferes with the amplification process in a traditional one-pot method reaction is solved, the detection sensitivity and specificity are improved, and a new technical means is provided for on-site rapid detection of the monkey pox virus.

The invention discloses Bxb1 recombinase with improved activity and application of the Bxb1 recombinase. The invention provides a recombinase, and the amino acid sequence of the recombinase comprises one or more amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO: 1. The recombinase provided by the invention can act on large-fragment DNA in a genome, so that the activity of a recombinase genome editing system is improved, and the application scene and selection of recombinase genome editing are enriched.

The invention discloses a construction method for improving gene editing efficiency of a recombinant trichoderma reesei engineering strain, and belongs to the technical field of gene editing. In the research, the T.reesei CJ2095 is taken as an original strain, the gene ku80 is knocked out, and the gene editing efficiency is improved to 7.14% from 1.04% of that of a control strain. In order to realize the recycling of the screening marker of the orotidine-5 '-phosphate decarboxylase gene pyr4, the gene pyr4 is knocked out. In addition, homologous recombinase rad51 and rad52 genes of a T.reesei endogenesis are respectively overexpressed, and the overexpressed gene rad52, namely the homologous recombination efficiency of the engineering strain T.reesei CJ2095 delta ku80 delta pyr4: rad52, can be improved to 43.75%, which is 42 times of that of an original strain.

The invention discloses an artificially optimized cDNA (complementary deoxyribonucleic acid)-based high-expression trametes versicolor chitin endonuclease gene, a recombinant vector, recombinant bacteria, recombinant enzyme and a preparation method thereof. The optimized trametes versicolor chitin endonuclease gene, such as a sequence as shown in SEQ ID NO.1, is constructed into a pichia pastoris expression vector and then converted into a pichia pastoris expression strain; according to the method, recombinant bacteria expressing active trametes versicolor chitin endonuclease are obtained through high-Zeocin-resistance YPD plate screening, high-expression pichia pastoris transformants are further screened from high-Zeocin-resistance transformants, finally fermentation is carried out in a bioreactor, obtained supernate contains a large amount of recombinant active trametes versicolor chitin endonuclease, and the recombinant active trametes versicolor chitin endonuclease can be obtained under the high-density fermentation condition. The expression level of the recombinant trametes versicolor chitin endonuclease reaches 3.1 g / L, and the trametes versicolor chitin endonuclease is efficiently purified through ammonia sulfate fractional precipitation and ion exchange. The recombinase can hydrolyze colloidal chitin to generate chitin oligosaccharide, and has important application value and wide application prospect in the aspects of preparation of functional chitosan oligosaccharide, antibiosis and anti-tumor, agricultural and biological control, environmental protection, industrial catalysis and the like.

The application discloses a carbonyl reductase mutant and application thereof in preparation of (R)-8-chloro-6-hydroxyoctanoate compounds. The application relates to a mutant of carbonyl reductase with improved catalytic performance and stereoselectivity after rational molecular modification, a recombinant expression plasmid containing the mutant gene of the enzyme, a recombinant expression transformant, a preparation method of the recombinant enzyme, and application of the recombinant enzyme or the recombinant cell as a catalyst in asymmetric reduction of 8-chloro-6-carbonyloctanoate compounds (I) to prepare (R)-8-chloro-6-hydroxyoctanoate compounds (II), in particular, in an intermediate (R)-8-chloro-6-hydroxyoctanoate methyl ester of thioctic acid. The disclosed carbonyl reductase SsCR mutant has the advantages of high reaction efficiency and high catalytic activity, and exhibits the advantages of high substrate concentration, low catalyst consumption and short reaction time in the process of catalytic preparation of the compound (II).

The invention discloses polyethylene glycol terephthalate hydrolase PmPETase and application thereof.The hydrolase PmPETase is protein with the amino acid sequence shown in SEQ ID NO.1. An expression system suitable for the PmPETase is constructed, recombinase protein with catalytic activity is obtained, and the polyethylene glycol terephthalate hydrolase PmPETase can be used for preparing the polyethylene glycol terephthalate hydrolase PmPETase. The substrate specificity, the catalytic property and the potential plastic degradation capacity of the PET degrading enzyme are comprehensively analyzed, and a new theoretical basis and application reference are provided for development and utilization of the PET degrading enzyme and a high polymer material green treatment technology.

The invention relates to the technical field of water pollution and water body odor substance detection, in particular to a primer group, a kit and a method for detecting cyanobacteria generating 2-methylisoborneol in a water body based on a recombinase-mediated isothermal nucleic acid amplification (RAA) technology. The primer group is designed based on a mic gene conserved region of the 2-methylisoborneol-producing cyanobacteria, and comprises a forward primer, a reverse primer and a specific probe; the 5'end of the reverse primer is modified with a fluorescent marker, the 5 'end of the probe is modified with biotin, the 3' end of the probe is modified with a phosphorylation group, and nuclease cleavage sites are contained in the probe. The primer group disclosed by the invention can effectively amplify the mic gene of the 2-methylisoborneol, has relatively high specificity and sensitivity, has no cross reaction with cyanobacteria which does not generate the 2-methylisoborneol, and can be combined with lateral flow immunochromatography to be used for on-site rapid detection of the 2-methylisoborneol; the method is of great significance in early warning of excessive content of 2-methylisoborneol in the water body.

The present disclosure relates to production of cis-3-hexenol from unsaturated fatty acid, such as alpha linoleic acid and linoleic acid. The process of the present disclosure involves combinatorial and sequential action of recombinant enzymes, such as LOX, HPL and ADH for the production of cis-3-hexenol.

The present invention refers to a recombinant Ten Eleven Translocation 3 (TET3) enzyme, a nucleic acid molecule encoding the TET3 enzyme, a vector comprising the nucleic acid molecule and a host cell transformed or transfected with the vector, the use of the recombinant TET3 enzyme for sequencing of a nucleic acid substrate, a method for sequencing a nucleic acid substrate and a reagent for sequencing a nucleic acid substrate.

The invention relates to screening methods. In particular, it relates to a method of identifying a functional TCR from a library of particles, which displays a plurality of different T cell receptors (TCRs). The method may comprise a) exposing the library of particles to a target antigen to identify TCRs that bind to the target antigen, b) transfecting a plurality of T cells with nucleic acid encoding TCRs identified in step (a), c) exposing the plurality of transfected cells to the target antigen, and d) selecting cells having TCR activity in the presence of the target antigen. Transfection of the T cells is such that each T cell comprises a nucleic acid encoding a single TCR from the library of particles, the nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and the TCR is in a single chain format and comprises an alpha chain variable domain, a beta cain variable domain and a constant domain. The T cells do not express endogenous TCR. A T-cell comprising a recombinase mediated landing pad, wherein the T cell constitutively expresses TCR constant domain and does not express endogenous TCR, is also claimed.