190 results about "Off targets" patented technology

This invention provides methods for determining drug specificity, therapeutic index and effective doses for individual patients. According to the methods of the invention, graded levels of drug are applied to a biological sample or a patient. A plurality of cellular constituents are measured to determine the activity of the drug on a target pathway and at least one off-target pathway. A drug specificity is determined by comparing the target and off target activities of the drug. A therapeutic concentration (or dose) is defined as a concentration (or dose) of the drug that induces certain response in the target pathway. A toxic concentration (or dose) is defined as a concentration (or dose) of the drug that induces certain response in the off target pathway. Therapeutic index is the ratio of the toxic concentration over therapeutic concentration. Methods are also provided to determine an effective dose of a drug for a patient by measuring the activity of the drug on the particular patient.

Methods and compositions for performing RNA interference with decreased off-target effects are provided The methods and compositions permit effective and efficient applications of RNA interference to applications such as diagnostics and therapeutics through the use of modifications to the siRNA. Uniquely modified siRNAs have been developed that reduce off-target effects incurred in gene-silencing. The modifications comprise 2′-O-alkyl or mismatch modification(s) at specific positions on the sense and/or antisense strands.

The invention is directed to modified oligonucleotide compositions and methods for selectively reducing unwanted nucleic acid contaminants and enriching for desired nucleic acid targets from complex genomic nucleic acid mixtures for sequencing applications. The modified oligonucleotide compositions include one or more modified groups that increase the T_m of the resultant oligonucleotide composition.

Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with improved specificity and methods for generating and using such TALEs.

The present invention is directed to pharmaceutical and therapeutic compositions which comprise RNA complexes comprising an antisense strand and a discontinued passenger strand capable of regulating gene expression. The use of a discontinued passenger strand reduces off target effects of the RNA complexes and also has other advantages.

The invention discloses a method for breeding rmnd5b (required for meiotic nuclear division 5homolog B) gene deletion type zebra fish through gene knockout and belongs to the field of gene knockout. According to the method, construction of a gRNA expression vector and gRNA in-vitro synthesis are performed through design of a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) gene knockout target site, micro-injection is performed on an embryo of the zebra fish, the effectiveness of the target site is detected, tail cutting identification is performed, TA cloning is performed on a target sequence, plasmids are subjected to Sanger sequencing, an F1 generation of heritable zebra fish mutants is obtained, the same mutant female fish and male fish are picked from mutants of the F1 generation, hybridization is performed, an F2 generation of the zebra fish mutants is obtained, F2 generation homozygote is picked from the F2 generation of the zebra fishmutants, F3 generation pure-line inheritance is performed, and an rmnd5b gene deletion type zebra fish strain is obtained. The method is lower in off-target rate and has good medical research value inresearch of the correlation between rmnd5b gene deletion and development of other organs.

The invention relates to the technical field of gene knockout, and particularly discloses a method for breeding a tcf25 gene deletion type zebra fish through gene knockout. The method comprises the steps of through a CRISPR/Cas9 gene editing technology, designing an appropriate targeting gene locus on a tcf25 gene of the zebra fish, synthesizing in vitro to obtain specific sgRNA and Cas9-mRNA, microinjecting into a cell of the zebra fish, and after culturing an embryo for 60h, selecting the embryo for carrying out genotyping, so that the effectiveness of the selected locus is verified. The method provided by the invention is lower in off-target rate, removes the tcf25 gene through interference, is beneficial to further revealing the whole process of cardiac morphogenesis and a molecular mechanism regulating the process through researching functions through a genetics means, and is of great importance on understanding a cardiac disease pathology and researching and developing a new therapeutic schedule medically.

The embodiment of the present invention provides a getting-off targeted reminding method and device. The method comprises the following steps: receiving at least one piece of vehicle usage informationof a passenger, wherein the vehicle usage information includes identification (ID) information and/or route information of the passenger, and the ID information includes the account, the mobile phonenumber, the photograph and/or the name of the passenger; detecting the vehicle environment and information of the boarding passenger, including detecting the seat position information of the passenger and detecting the luggage position information, and determining whether the passenger carries luggage or not; a calculation processing module receiving the detected data and matching the data with the vehicle usage information to determine whether to remind the passenger or not according to needs; and reminding the passenger getting off, reminding the passenger not getting off, or reminding thepassenger missing the luggage or taking the wrong luggage according to an instruction of the calculation processing module. According to the invention, the seat, the luggage position and the ID of thepassenger are bond, thereby being capable of reminding the passenger getting off when the passenger needs to get off.

The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

The present invention relates to a novel siRNA structure and the use thereof. More particularly, the invention relates to a double-stranded small interfering RNA molecule (siRNA molecule) comprising a 19-21 nucleotide (nt) antisense strand and a 15-19 nt sense strand having a sequence complementary to the antisense sequence, wherein the 5′ end of the antisense strand has a blunt end and the 3′ end of the antisense strand has an overhang, and to a method for silencing the expression of a target gene using the siRNA molecule.

This invention relates to reagents and methods for increasing specificity and efficiency of genome editing by CRISPR associated (Cas) protein systems, more particularly by Cas:guide RNA complexes, by blocking off-target nucleic acids from cleavage by Cas:guide RNA complexes.

The invention discloses a gRNA sequence. The sequence is capable of editing a DNA sequence in the manner of taking a hepatitis B viral genome specific locus as a target sequence. The invention also discloses a CRISPR-SaCas9 system containing the gRNA sequence and a recombinant adeno-associated virus packaged with the system. The system and the packaging virus show higher HBV scavenging activity in cells and in transgenic mice body. On the 38th day after twice continuous high dose injection, the contents of HBsAg and HBeAg in experimental group mice serum, compared with the control group, are respectively reduced by 62.96+/-7.59% and 65.18+/-3.08%; the HBV DNA content in the serum, compared with the control group, is reduced by 92.82+/-3.67%; the liver and other visceral organs of the experimental mice are all free from any pathologic change, the off-target effect is also not detected and the application prospects of the gRNA sequence and the corresponding CRISPR-SaCas9 system provided by the invention in preparing the hepatitis B therapeutics are shown.

The invention relates to a method for achieving HMGCR gene knockout based on the CRISPR/Cas9 technology. The method is characterized in that two CRISPR/Cas9 target sequence aiming at the HMGCR gene isdesigned, a gRNA single chain is synthesized in vitro, annealing is performed to obtain two gRNA double-chain DNA target insertion fragments, the insertion fragments are inserted into PX459 (pSpCas9(BB)-2A-Puro)V2.0 vectors to obtain the two different-locus plasmids of the target HMGCR gene; the two plasmids are transfected into PK15 cells, puromycin is used to process the cells, the processed cell genome DNA is extracted to perform PCR amplification, the PCR product is denatured, annealing is performed, and then T7E1 is used to perform HMGCR gene knockout identification. The method has the advantages that method can be used for analyzing the expression conditions of sequence and mRNA after the HMGCR gene knockout, whether an off-target phenomenon exists or not can be verified by using amethod combining PCR and T7E1 enzyme treatment, and accordingly the specificity based on target sequence HMGCR-gRNA can be determined; the method is applicable to cell and animal models to achieve fixed-point HMGCR gene knockout, has a reference value to the knockout of other genes, and is good in effect, simple, economical, short in time and the like.

The invention provides a highly specific CRISPR genome editing system. The gRNA of a target Cas9 gene is introduced into a CRISPR/Cas9 genome editing system, when (or after) target cell genome editingis performing, the gRNA of the target Cas9 gene can mediate the cutting of Cas9 gene, thus the expression time of the Cas9 gene is prominently reduced, the off-target effect caused by long term expression of Cas9 in a CRISPR system is reduced, and the targeting specificity is enhanced.

This invention encompasses compounds, structures, compositions and methods for therapeutic guide molecules that direct CRISPR gene editing. A guide molecule for directing gene editing can be allele selective, or disease allele selective, and can exhibit reduced off target activity. A guide molecule can be composed of monomers, including UNA monomers, nucleic acid monomers, and modified nucleotides, wherein the compound is targeted to a genomic DNA. The guide molecules of this invention can be used as active ingredients for editing or disrupting a gene in vitro, ex vivo, or in vivo.

The invention provides a method for detecting the off-target effect of an adenine base editor system based on whole genome sequencing, and its application in gene editing, The adenine base editor system comprises a TadA:TadA*:Cas9 fusion gene and gRNA. The system can catalyze the efficient replacement of adenine (A) at a target site with guanine (G), and has a broad application prospect in human disease gene editing therapy and disease model construction. The first method for detecting the off-target effect of the ABE system in a whole genome range, called EndoV-seq method for short, is developed in the invention. The EndoV-seq method provided by the invention has a broad application prospect in the field of gene editing, especially gene editing therapy.

The present invention provides oligomers that binds RNA that consists of a lower affinity region and a higher affinity region, wherein the monomers in the higher affinity region increases the melting temperature (i.e. affinity) of the oligomer base paired to RNA more than the monomers used in the lower affinity region, wherein said increase in melting temperature is relatively to the alternative use of DNA monomers of the same (base) sequence. The oligomers of the invention are useful for binding to target RNA such as mRNA and non-coding RNA and have the advantage that they will be less prone to off-target binding via the lower affinity region than via the higher affinity region.

This invention provides principles, methods and compositions for ascertaining the mechanism of action of pharmacologically important compounds in the context of network biology, across the entire scope of the complex pathways of living cells. Importantly, the principles, methods and compositions provided allow a rapid assessment of the on-pathway and off-pathway effects of lead compounds and drug candidates in living cells, and comparisons of lead compounds with well-characterized drugs and toxicants to identify patterns associated with efficacy and toxicity. The invention will be useful in improving the drug discovery process, in particular by identifying drug leads with desired safety and efficacy and in effecting early attrition of compounds with potential adverse effects in man.

Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5′ nucleotide of the target is specific for thymine (T). TALE domains with alternative 5′ nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities. Some aspects of this disclosure provide engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with altered 5′ nucleotide specificities of target sequences. Engineered TALEs that target ATM with greater specificity are also provided.

In principle, protein-based biotherapeutics offers a way to control biochemical processes in living cells under non-steady state conditions and with fewer off-target effects than conventional small molecule therapeutics. However, systemic protein delivery in vivo has been proven difficult due to poor tissue penetration and rapid clearance. Protein transduction exploits the ability of some cell-penetrating peptide (CPP) sequences to enhance the uptake of proteins and other macromolecules by mammalian cells. Previously developed hydrophobic CPPs, named membrane translocating sequence (MTS), membrane translocating motif (MTM) and macromolecule transduction domain (MTD), are able to deliver biologically active proteins into a variety of cells and tissues. Various cargo proteins fused to these CPPs have been used to test the functional and/or therapeutic efficacy of protein transduction. The recombinant proteins consisting of suppressor of cytokine signaling 3 (CP-SOCS3) protein fused to the fibroblast growth factor (FGF) 4-derived MTM were developed to inhibit inflammation and apoptosis. However, CP-SOCS3 fusion proteins expressed in bacteria cells were hard to be purified in soluble form. To address these critical limitations, CPP sequences called advanced MTDs (aMTDs) have been developed in this art. This is accomplished by (i) analyzing previous developed hydrophobic CPP sequences to identify specific critical factors (CFs) that affect intracellular delivery potential and (ii) constructing artificial aMTD sequences satisfied for each critical factor. In addition, solubilization domains (SDs) have been incorporated into the aMTD-fused SOCS3 recombinant proteins to enhance solubility with corresponding increases in protein yield and cell-/tissue-permeability. These recombinant SOCS3 proteins fused to aMTD/SD having much higher solubility/yield and cell-/tissue-permeability have been named as improved cell-permeable SOCS3 (iCP-SOCS3) proteins. Previously developed CP-SOCS3 proteins fused to MTM were only tested or used as anti-inflammatory agents to treat acute liver injury. In the present art, iCP-SOCS3 proteins have been tested for use as anti-cancer agents in the treatment of various cancers likes gastric, colorectal and breast cancer, and glioblastoma. Since SOCS3 is frequently deleted in and loss of SOCS3 in tumors promotes resistance to apoptosis and proliferation, we reasoned that iCP-SOCS3 could be used as a protein-based intracellular replacement therapy for the treatment of various cancers. The results demonstrated in this art support the reasoning: treatment of cancer cells with iCP-SOCS3 results in reduced cancer cell viability, enhanced apoptosis of solid tumors including gastric, colorectal and breast cancer, and glioblastoma and loss of cell migration/invasion potential. Furthermore, iCP-SOCS3 inhibits the growth of gastric and colorectal tumors in a subcutaneous xenografts model. In the present invention with iCP-SOCS3, where SOCS3 is fused to an empirically determined combination of newly developed aMTD and customized SD, macromolecule intracellular transduction technology (MITT) enabled by the advanced MTDs may provide novel protein therapy against various tumors such as gastric cancer, colorectal cancer, glioblastoma, and breast cancer.