145results about How to "Reduce off-target effects" patented technology

The invention relates to a double-stranded RNA (dsRNA) molecule comprising between about 15 base pairs and about 40 base pairs, wherein at least one ribonucleotide of the dsRNA is a 5′-methyl-pyrimidine, and a method of using such modified dsRNA molecule to increase stability of RNA when in contact with a biological sample.

The invention discloses a CRISPR-Cas9 system used for assembling DNA and a DNA assembly method. The CRISPR-Cas9 system includes the following parts: a plasmid used for expressing Cas9 gene, a first guide RNA, and / or a plasmid used for expressing the first guide RNA, wherein the first guide RNA has a CRISPR site. The CRISPR site is combined with a first reporter gene carried by a semisynthetic chromosome used for assembling according to base complementation pairing rule. The CRISPR-Cas9 system has advantages of high replacement success rate of homologous recombination, less species of guide RNA which needs to design and use, less harmful effect subjected by the genomic sequence, and low off-target rate.

The invention relates to a double-stranded RNA (dsRNA) molecule comprising between about 15 base pairs and about 40 base pairs, at least one 5′-methyl-pyrimidine and at least one 2′—O-methyl ribonucleotide, and a methods of using such modified dsRNA molecule to increase stability of a siRNA molecule when in contact with a biological sample, to reduce off-target effects and to reduce interferon responsiveness (IFN) of the siRNA molecule.

The invention aims at the technical defects of off-target effects of an existing CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated protein 9) gene editing technology to carry out site-directed mutation of an amino acid site on a site combined with a DNA (Deoxyribonucleic Acid) sequence of Cas9 protein to obtain the Cas9 protein with low off-target efficiency,so as to realize a gene editing unction with higher targeting performance and higher efficiency. Meanwhile, the invention further provides a method for carrying out HEK293T cell gene editing by adopting a gene editing method.

The present invention generally provides vectors, compositions, and methods of using the same for gene therapy.

The invention discloses a goat TLR4 gene knock-out vector and a construction method thereof. The construction method comprises the following steps: firstly, designing an sgRNA fragment of the TLR4 gene by adopting a CRISPR / cas9 system, synthesizing an sgRNA nucleotide sequence, constructing and simultaneously expressing the sgRNA and plasmid PYSY-sgRNA of Cas9 D10A, connecting and transforming to an Escherichia coli DH5 alpha competent cell, and verifying the transformant; and judging and proving by enzyme digestion and sequencing that the TLR4 gene knock-out vector is constructed correctly. The invention adopts the CRISPR / cas9 for constructing the vector, and provides a theoretical basis for subsequently acquiring a goat TLR4 gene deletion type alveolar epithelial cell system, and studying the immune response molecular mechanism of mycoplasma pneumonia infection.

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.

A hemi-sulfate salt of the structure:to treat a host infected with hepatitis C, as well as pharmaceutical compositions and dosage forms, including solid dosage forms, thereof.

The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention provides Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them, and also provides complexes for nucleic acid editing (for example, gene or genome editing) comprising the above-mentioned proteins or nucleic acid molecules. Objects and compositions, and methods for nucleic acid editing (eg, gene or genome editing) comprising the above proteins.

The invention belongs to the field of nucleic acid editing, and particularly relates to the technical field of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In particular, the present invention provides Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them, and also provides complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising the aforementioned proteins or nucleic acid molecules, and methods for nucleic acid editing (e.g., gene or genome editing) comprising the aforementioned proteins.

The invention relates to a kit and particularly relates to a kit for editing or repairing human hemoglobin gene. The kit comprises sgRNA of specific target HBB gene or a carrier containing the nucleotide sequence of the sgRNA; and / or modified Cas9 enzyme or a sequence encoding the modified Cas9 enzyme or a carrier containing the sequence encoding the modified Cas9 enzyme; and / or a donor gene sequence for repairing the HBB gene of beta-thalassemia or a carrier containing the donor gene sequence. The invention also provides an application of the kit in cutting or repairing the HBB gene, especially an application in repairing the HBB gene of autologous hematopoietic stem cells of a patient with beta-thalassemia. The kit provided by the invention has the advantages that the targeting capability of the target HBB gene is strong, the cutting efficiency of the HBB gene is high, the off-target efficiency is low, the HBB gene can be effectively repaired and the prospect in clinical study and treatment application is wide.

The present invention discloses a novel mitochondrial genome editing tool, and belongs to the field of genome engineering. According to the prevent invention, the constructed mtCRISPR / Cas9 system mainly comprises two parts such as gRNA entering mitochondria and Cas9 nuclease localized in mitochondria, wherein the constructed mt-gRNA has two forms, the one mt-gRNA comprises a RNA mitochondrial localizing guide sequence, a targeting sequence and a gRNA skeleton sequence, the other mt-gRNA comprises a RNA mitochondrial localizing guide sequence, any one tRNA sequence encoded by mitochondrial or other additional spacer sequences, a targeting sequence and a gRNA skeleton sequence, the obtained combined material acts on the mitochondrial genome to break the target sequence in a targeted manner after the mt-gRNA and the mtCas9 nuclease are combined, and the action efficiency of the second mt-gRNA action is high than the action efficiency of the mt-gRNA; and the results verify that the constructed mtCRISPR / Cas9 system has characteristics of high efficiency and strong specificity.

Compositions and methods for down modulating expression of target nucleic acids are disclosed. This invention relates to the fields of medicine, drug development and modulation of target nucleic acid expression. More specifically, the invention provides compositions and methods of use thereof that facilitate the modulation of target nucleic acid expression using novel oligonucleotide based drugs that act through an inhibitory RNA (RNAi) mechanism of action.

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 discloses an oligonucleotide molecule used for inhibiting expression of mRNA of a target gene and a composition set thereof, and provides siRNA which is composed a positive-sense strand which is composed of 19-27 nucleotides and a negative-sense strand being reverse-complementary with the positive-sense strand. In the positive-sense strand, both 5-9 continuous nucleotides from a 5'-terminal and 5-9 continuous nucleotides from a 3'-terminal are 2'-ribose modified nucleotides. The siRNA molecular mixture can influence expression of target genes in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and 90% cells, and is at least 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95% in inhibition ratio.

The invention belongs to the field of hepatitis B medicine, and particularly relates to a siRNA composition for treating viral hepatitis B. The composition comprises a siRNA sequence and other components, wherein the siRNA sequence is ID No. 267; the other components include 0.5-3 parts by weight of cationic lipid, 2-8 parts by weight of T2C1 compound, 0.5-3 parts by weight of phospholipid, 0-2 parts by weight of cholesterol and 2-10 parts by weight of lipid-polyethylene glycol. Under the situation of achieving the same effect, the injection dosage of a siRNA preparation is much lower than the dosage of mouse medicine in the prior art.

The invention relates to biological technology, specifically relates to a chimeric protein pAgoE, a construction method and applications thereof, and at the same time, relates to a chimeric protein pAgoE using a guide, a construction method and applications thereof, and a chimeric protein, which is based on the chimeric protein pAgoE and has a genome targeting editing function. The chimeric protein pAgoE contains a pAgo structural domain with a genome targeting positioning function and an effect structural domain E, which is connected to the pAgo structural domain and has a genome cutting or modifying activity. The pAgo structural domain with a genome targeting positioning function and the effect structural domain E with a genome cutting or modifying activity are fused to establish the chimeric protein pAgoE; the chimeric protein pAgoE maintains the original targeting binding performance of the genome (DNA) having a targeting positioning function in the pAgo structural domain and the genome catalytic or modifying performance of the effect structural domain E at the same time, and an sufficient space is provided for the structural domain E to fully exert the effect.

The invention discloses an MCIR (MelanoCortin 1 Receptor) gene carrier and a construction method thereof. The MCIR gene carrier comprises MC1R-neo and MC1R-GFP (Green Fluorescent Protein), wherein the nucleotide sequence of the MC1R-neo is SEQ ID NO:1, and the nucleotide sequence of the MC1R-GFP is SEQ ID NO:2. A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system is adopted to construct an MC1R gene knock-out vector, and only a sgRNA (Ribonucleic Acid) of which the length is about 20bp needs to be designed by aiming at a gene knock-out point to be connected with a universal Cas9 gene. Therefore, compared with traditional gene knock-out technologies including ZFNs (Zinc finger nuclease), TALENs (Transcription Activator-Like Effector Nucleases) and the like, the construction method disclosed by the invention adopting the CRISPR / Cas9 to construct the gene knock-out vector is simpler and more convenient and is more convenient to be further popularized and applied to subsequent experiments.

This invention provides UNA oligomers for gene silencing with reduced off-target effects. The UNA oligomers can have a first strand and a second strand, each of the strands being 19-29 monomers in length, the monomers being UNA monomers and various nucleic acid monomers. Embodiments include pharmaceutical compositions and methods for treating or preventing TTR-related amyloidosis with reduced off-target effects by administering a UNA oligomer to a subject.

The present disclosure provides a siRNA for inhibiting the expression of apolipoprotein C3 (ApoC3) gene, and a pharmaceutical composition and a conjugate comprising the siRNA; wherein each nucleotide in the siRNA is independently a modified nucleotide, and the siRNA comprises a sense strand and an antisense strand; the sense strand comprises a nucleotide sequence A, the nucleotide sequence A having the same length as the nucleotide sequence as represented by SEQ ID NO:1 with no more than 3 nucleotide differences; the antisense strand comprises a nucleotide sequence B, the nucleotide sequence B having the same length as the nucleotide sequence as represented by SEQ ID NO:2 with no more than 3 nucleotide differences.

The invention provides a chemically modified double-strand small interfering RNA (siRNA) molecule and its preparation and application methods. Chemically modified nucleotide is introduced into the 14th site or / and 16th site beginning from 5' terminal of the siRNA molecular sense strand, thereby reducing the off-target effect caused by the modified siRNA molecular sense strand and enhancing specificity of the modified siRNA molecule for silence of target gene expression.

The present invention relates to Argonaute-nucleic acid sequence component systems, methods and compositions for sequence manipulation. Specifically, the present invention relates to Argonaute, Argonaute-nucleic acid sequence component composition, Argonaute-nucleic acid sequence component sequence manipulation system capable of cleaving target nucleotide sequences with the help of nucleic acid strands, and the use of said Argonaute, Argonaute-nucleic acid Sequence Component Compositions and Argonaute-Nucleic Acid Sequence Component Sequence Manipulation System Methods for Manipulating Sequences. The Argonaute of the present invention can cut target nucleotide sequences with the help of nucleic acid chains at normal temperature, the cutting efficiency is not lower than 20%, and the optimal salt concentration is 100-200 mmol / L.

Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.

Provided is a method for achieving gene knockout by utilizing a base editing technique and application thereof. The gene knockout method comprises the steps that a 20bp-NGG target sequence in the encoding area which genes are to be knocked out is selected and is made including a complete target codon CAA, CAG or CGA; an SgRNA sequence is utilized to localize a base editing system to the target sequence so as to change a single base C in the target codon into T, and thus a termination codon TAA, TAG or TGA is introduced to achieve gene knockout, wherein the target single base C is located at the position 3-8 of the target sequence, and the sgRNA sequence is a 20bp sequence complementary with the target sequence. The method still has higher activity in primary cells or hypermethylated regions, so the knockout method is more efficient, more accurate, less missed and wider in application range, and is a more advanced method for gene knockout.

The invention provides a cell strain capable of reducing replicability adenovirus production and a construction method and an application thereof. The cell strain HEK 293.CS capable of reducing replicability adenovirus production is a safe adenovirus production cell line by knocking out of a gene fragment in HEK293 which is homologous with an Ad5 adenovirus E1 gene, and simultaneously providing template plasmid for replacing the gene fragment to a non-homologous sequence which is capable of stabilizing the E1 gene expression. Compared with un-reconstructed HEK293 cell strain, the growth capability and virus production capability of HEK 293.CS are not decreased, but detectable RCA is not produced; the cell strain can be used for large-scale cultivation of recombinant human adenovirus type 5, and the RCA production probability during medicine manufacture such as vaccines and antibodies is reduced.

The invention relates to siRNA for inhibiting expression of a CTGF gene. The siRNA contains a positive-sense strand and an antisense strand, wherein the positive-sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are both 19 nucleotides in length, and the nucleotide sequence II at least partially and mutually complements a sequence which is 19 nucleotides in length in a region from site No. 700 to site No. 1050 of CTGF mRNA. From a terminal 5' to a terminal 3', nucleotides at sites No.7, No. 8 and No. 9 of the nucleotide sequence I are fluoro-modified nucleotides, and nucleotides at sites No. 2, No. 6, No. 14 and No. 16 of the nucleotide sequence II are fluoro-modified nucleotides.The invention further provides a pharmaceutical composition containing the siRNA. The siRNA and the pharmaceutical composition containing the siRNA can be used for treating or improving diseases relevant with the expression of the CTGF gene.

The present invention provides immunoresponsive cells, including T cells, cytotoxic T cells, regulatory T cells, and Natural Killer (NK) cells, expressing an antigen recognizing receptor and an inhibitory chimeric antigen receptor (iCAR). Methods of using the immunoresponsive cell include those for the treatment of neoplasia and other pathologies where an increase in an antigen-specific immune response is desired.

The invention discloses a NK (Natural Killer) cell with a chimeric CEA (Carcino-Embryonic Antigen) antigen receptor and a preparation method and applications thereof. The NK cell with the chimeric CEAantigen receptor, which is disclosed by the invention, is obtained by expressing the chimeric CEA antigen receptor in a NK cell. The NK cell with the chimeric CEA antigen receptor has the following characteristics that: 1, tumor cells can be efficiently and specifically killed; 2, while the CEA-CAR (Chimeric Antigen Receptor) is expressed, the regulatory mechanism of the NK cell per se is not affected, that is, the ability to inhibit the effect on homologous normal cells is not affected; and 3, after in vitro expansion of lymphocytes, the NK cell accounts for more than 90% of the harvested total cells and the CEA-CAR-NK accounts for more than 34% of the harvested total cells. The NK cell with the chimeric CEA antigen receptor, which is disclosed by the invention, plays an important role in clinical treatment of various solid tumors when serving as a biological agent.

The invention belongs to the field of genetic engineering and particularly relates to a method for knocking out a pseudorabies virus TK gene by using dual sgRNA and application of the method. According to the method, a dual-locus knockout method is employed, two sgRNA sequences are designed on the TK gene, two loci are cut in a pointed manner, and thus, the TK gene can be efficiently knocked out. The method for knocking out the pseudorabies virus TK gene mainly comprises the procedures of sgRNA designing, Cas9 vector-sgRNA expression vector constructing, donor vector constructing, plasmid transfection, cre enzyme vector transfection and recombinant virus purification. Compared with the traditional methods, the method disclosed by the invention has the advantages of high accuracy and low target missing effect.