44 results about "Dna cleavage" patented technology

The invention provides Cas12a mutants from Murraya bovis AAX08, and methods of using the Cas12a mutants. The Mb2Cas12a mutant comprises a single amino acid substitution, a plurality of amino acid substitutions, a structural domain exchange, or all of the above. Compared with a wild type moraxella bovine eye AAX08Cas12a enzyme, the mutants have enhanced DNA (Deoxyribose Nucleic Acid) lysis activity in plants.

The present disclosure relates to aryl diazonium compounds and methods of cleaving DNA using the diazonium compounds as described herein. Further, the present disclosure relates to said aryl diazonium compounds conjugated to additional compounds as described herein.

Systems and kits are disclosed herein for genetic engineering (such as for DNA cleavage and gene-editing), which include catalytic nucleic acids and catalytic nucleic acid-assisting reagents. Methods of genetic engineering are also described, in which both catalytic nucleic acid-assisting reagents and catalytic nucleic acids are specific for a target site, thus, providing high-fidelity genetic engineering.

The present disclosure concerns polynucleotides and amino acids of Acidaminococcussp. Casl2a (Cpfl) and methods for their use for genome editing in eukaryotic cells. 20 24 20 14 49 05 M ar 2 02 4 A B S T R A C T 2 0 2 4 2 0 1 4 4 9 0 5 M a r 2 0 2 4

The present invention relates to modified gRNA molecules, compositions and methods for site specific gene editing and genomic modification, such as DNA cleavage, and gene activation or repression. The present modified guide RNAs have modified secondary structure (e.g., long upper stem and a modified hairpin structure), which target the target DNA sequence specifically with reduced off-target activity.

Compositions and methods are provided that include engineered anti-CRISPR (Acr) proteins that can achieve a balance in which the Cas protein retains activity to perform targeting functions at a target nucleic acid (e.g., DNA cleavage for gene editing applications), but is inhibited by the subject Acr protein to a degree to reduce off-target activity, such that the subject Acr protein may not inhibit off-target activity. Thus, the ratio of target to off-target nucleic acid targeting events is increased. In some cases, the subject compositions (e.g., subject systems) include a CRISPR nuclease or a nucleic acid encoding the same. Methods of using the subject Acr polypeptides with CRISPR nuclease enzymes to achieve target nucleic acid modification are provided.

The present disclosure concerns polynucleotides and amino acids of Acidaminococcus sp. Cas12a (Cpf1) and methods for their use for genome editing in eukaryotic cells.

The invention particularly relates to preparation of a CRISPR (clustered regularly interspaced short palindromic repeats)-immune combined small molecule detection probe as well as a related detection method and application thereof. The preparation method of the probe mainly adopts a chemical and biological coupling method, and the dsDNA-antibody probe is prepared by covalently coupling the dsDNA of which the 5'end is modified to NH2 and a monoclonal antibody by using a bifunctional cross-linking agent 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid group succinimide ester sodium salt (Sulfo-SMCC) and N-succinimido-S-acetylthioacetate (SATA). Secondly, according to the detection method, a dsDNA-antibody probe is taken as a bridge to combine an immune system and a CRISPR / Cas12a system, the specific recognition capability of an antibody to small molecules is utilized, signal amplification is carried out by virtue of RPA nucleic acid amplification, and fluorescence signals are generated by virtue of cis-cleavage activity and / or trans-single-chain DNA cleavage activity of CRISPR / Cas12a, so that conversion from immune reaction signals to high-intensity fluorescence signals is realized, and the detection sensitivity is high. And a CRISPR-immune combined small molecule detection system is established. The detection method has the advantages of simplicity in operation, high specificity, extremely high sensitivity, stable signal and the like.

Disclosed herein are compositions and methods for treatment and prevention of low back pain. The compositions include vectors comprising nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs and a RNA-directed nuclease. The methods include modulating expression of one or more genes in a cell using the compositions, introducing a CRISPR-Cas system into a cell comprising one or more vectors comprising the compositions, including site-specific DNA cleavage in a cell, and treating a subject having lower back pain, and lower back pain caused by degenerative disc disease using the compositions disclosed herein.

Bridge helix-modified variant Cas12a and Cas12b proteins having improved DNA cleavage selectivity in comparison to wild type versions of the Cas12a and Cas12b proteins, nucleic acids encoding the variant proteins, host cells containing the nucleic acids, and methods of their use. In particular embodiments, the at least one substitution or at least two substitutions are to proline. In certain embodiments, bridge helix-modified proteins are variants of the Cas12a proteins from Francisella tularensis novicida U112, Lachnospiraceae bacterium, and Acidaminococcus sp, and of the Cas12b protein from Alicyclobacillus acidoterrestris.

Provided are modified proteins that are functional in RNA-guided DNA cleavage. The proteins include modified IscBs protein that have a modification of the N-terminus or C-terminus, or both. The modifications include a truncation of a PLMP domain of the IscB protein, or a PLMP domain that is relocated to a position of the IscB protein that is not the N-terminus. The modified IscB protein can be provided as a component of a fusion protein. The modified IscB proteins are used with an ωRNA to modify a DNA substrate.

The present invention discloses an application of a chelating agent in inhibiting the catalytic activity of low-dimensional nanomaterials and DNA shearing. When the chelating agent is used to inhibit the catalytic activity of low-dimensional nanomaterials, the chelating agent is mixed with the low-dimensional nanomaterial, so that the electrons provided by the chelating agent interact with the dangling bonds of the defects of the low-dimensional nanomaterial to form covalent bonds, thereby inhibiting the catalytic activity of the low-dimensional nanomaterial. The application of the chelating agent in DNA shearing is to mix the chelating agent with the low-dimensional nanomaterial and then mix it with a DNA solution, so that the electrons provided by the chelating agent interact with the dangling bonds of the defects of the low-dimensional nanomaterial to form covalent bonds, thereby inhibiting the effect of the catalytic activity of the low-dimensional nanomaterial on DNA shearing. The present invention utilizes the chelating effect of the chelating agent on the defects of the low-dimensional nanomaterial to inhibit the catalytic activity of the defects of the low-dimensional nanomaterial, and utilizes this property to successfully inhibit the shearing effect of the low-dimensional nanomaterial on DNA, which is of great significance for the later development of related biological applications of nanozymes.

Here, the inventors developed a strategy to modify the chromatin status of HSPCs at the HBG promoters to reactivate HbF expression. In particular, the inventors used from 1 to 4 single guide RNA (sgRNA) molecules spanning from the -220 to the -20 region of the HBG promoters together with CRISPR-based epigenome editors, namely dCas9-CBPcore and Tet1-dCas9. The first editor is capable of inserting histone acetylation while the second editor is performing DNA demethylation. Both epigenetic marks are associated with active transcription and are present in fetal erythroid cells expressing HbF. This strategy allows the ex vivo modification of adult HSCs to recreate a fetal-like epigenetic context leading to HbF production. Moreover, it avoids the genotoxicity associated with classical genome editing tools relying on DNA cleavage. Thus, the present invention relates to a method of increasing the expression of gamma globin in a population of eukaryotic cells through epigenome editing.

Described herein are compositions and methods for treatment and prevention of low back pain. The compositions include vectors comprising nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs and a RNA-directed nuclease. The methods include modulating expression of one or more genes in a cell using said compositions, introducing a CRISPR-Cas system into a cell comprising one or more vectors comprising said compositions, inducing site-specific DNA cleavage in a cell, and treating a subject having lower back pain, and lower back pain caused by degenerative disc disease using the compositions disclosed herein.

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3 / Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule using an RNA-guided DNA endonuclease comprising at least one RNA sequence and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro or using a cassette containing a single repeat-spacer-repeat unit.

Provided are methods for treating cancer in a subject by selecting a therapeutic molecule after modifying cancer cells from the subject by knocking down gene function of one or more genes without targeted DNA cleavage of one or more genomic loci. Also provided herein are methods of perturbing gene function in one or more modified cancer cells from a subject without targeted DNA cleavage of one or more genomic loci. Also provided herein are CRISPRi systems for gene modulation and high titer CRISPRi lentiviral gene modulation solutions for knockdown of target gene function in primary cells.

The present invention relates to modified gRNA molecules, compositions, and methods for site-directed gene editing and genome modification, such as DNA cleavage and gene activation or repression. The modified guide RNA of the present invention has a modified secondary structure (e.g., a long upper stem and a modified hairpin structure) that specifically targets a target DNA sequence having reduced off-target activity.

The present disclosure relates to polynucleotides and amino acids of Acidaminococcus sp. Cas12a (Cpf1) and methods for their use for genome editing in eukaryotic cells.

Disclosed is a method for cleaving a target DNA to isolate a target DNA fragment sequence of interest, guided by a targeting oligonucleotide, followed by enrichment of the target DNA fragment sequence. The targeting oligonucleotide binds to the target DNA during DNA cleavage. After cleavage, the target DNA fragment sequence of interest is modified using a linking or polymerase extension method. The resulting target DNA fragment sequence of interest is enriched by exonuclease treatment.

A method for cleaving a target DNA to isolate a target DNA fragment sequence of interest, directed by a targeting oligonucleotide followed by enriching the target DNA fragment sequence is disclosed. The targeting oligonucleotide binds to the target DNA during DNA cleavage. After cleavage, the target DNA fragment sequence of interest is modified using either a ligation or a polymerase extension method. The resulting target DNA fragment sequence of interest is enriched by exonuclease treatment.

The application relates to a preparation method and application of an electrochemical sensor for sensitive detection of lead ions based on a metal nanocomposite. 2+ The introduction of the target Pb 2+ triggers the S-DNA cleavage of the DNAzyme to cut off a single-strand DNA, and simultaneously initiates the DNA Walker effect to obtain a large amount of single-strand DNA. The supernatant after magnetic separation is added dropwise on an electrode modified with HP1, after incubation, the single-strand DNA serves as an initiation chain to initiate the CHA signal amplification reaction, so that more and more signal probes are connected to the electrode surface, and the electrical signal in the system is enhanced. The quantitative analysis of lead ions is realized by using the change of the electrical signal corresponding to the change of the methylene blue concentration on the electrode surface before and after the addition of Pb 2+ The prepared electrochemical sensor has high sensitivity, a wide detection range, good selectivity and excellent practical application capability.

The present disclosure concerns polynucleotides and amino acids of Acidaminococcus sp. Cas12a (Cpf1) and methods for their use for genome editing in eukaryotic cells.

The application discloses an ICD inducer Ru(II) complex with a light-driven DNA breaking ability, a preparation method and application thereof, relates to the field of antitumor drugs, and the structural formula of the Ru(II) complex is [Ru(dip)2(L)](PF6)2. The Ru(II) complex can enter a cell nucleus to break DNA and activate immunogenic death mediated by the pyroptosis and cGAS-STING pathways by using photodynamic therapy, so that the antitumor immune effect of the body is improved. In the in-vivo experiment, the immune cells in the tumor microenvironment of the mice in the vaccine group are good, and the antitumor immune effect of the three injections of the vaccine is better. The results of H&E staining show that the vaccine does not cause obvious pathological damage to the main organs of the mice, and the vaccine has good biological safety.

The invention relates to the technical field of tumor immunotherapy, and particularly discloses a screening method of a cancer cell mitochondrial DNA (deoxyribonucleic acid) coding new antigen, mitochondrial DNA with higher purity is obtained by means of microdissection, linear DNA splitting and the like, and nuclear metastasis mitochondrial fragment interference in mitochondrial sequencing is overcome. On the basis, mitochondrial amplicon sequencing is used for identifying the base sequence change caused by mutation from the DNA level, multiple types of tumor vaccines including polypeptide vaccines, RNA vaccines and dendritic vaccines can be prepared according to requirements, and even the tumor vaccines can be used for TCR-T therapy. According to the screening method of the mitochondrial DNA coding new antigen and the application of the mitochondrial DNA coding new antigen in preparation of the vaccine, the feasibility of the mitochondrial coding new antigen and the effectiveness of the mitochondrial coding new antigen as a tumor vaccine target are accurately and comprehensively confirmed, and a transcription translation process and a screening mechanism of DNA-RNA-polypeptide are systematically constructed; and more diversified, personalized, safe and effective treatment strategies can be selected and designed for the patient.

This invention pertains to mutant Cas9 nucleic acids and proteins for use in CRISPR / Cas endonuclease systems, and their methods of use. In particular, the invention pertains to an isolated mutant Cas9 protein, wherein the isolated mutant Cas9 protein is active in a CRISPR / Cas endonuclease system, wherein the CRISPR / Cas endonuclease system displays increased on-target editing activity relative to a wild-type CRISPR / Cas endonuclease system. The invention also includes isolated nucleic acids encoding mutant Cas9 proteins, ribonucleoprotein complexes and CRISPR / Cas endonuclease systems having mutant Cas9 proteins that display increased on-target editing activity relative to a wild-type CRISPR / Cas endonuclease system.

This invention provides a method for detecting microRNA using thermophilic TtAgo protein based on cofactor activation of its mesophilic DNA cleavage activity. This invention expands the applicable temperature range of TtAgo by using a cofactor that extends the function of the extremely thermophilic TtAgo protein and activates its mesophilic DNA cleavage activity, thereby establishing a one-step closed-tube isothermal multiplex analysis method for microRNA based on TtAgo that is not limited by high temperatures.

To provide novel genome editing techniques that modify the nucleobase of a specific sequence of a gene without double-stranded DNA cleavage, or with single-strand cleavage, as well as to provide nucleic acid sequence recognition modules therefor, and to provide complexes of nucleobase converting enzymes.SOLUTION: Provided is a method comprising: contacting a complex, in which a nucleic acid sequence recognition module specifically binding to a target nucleotide sequence in a selected double-stranded DNA and a nucleobase converting enzyme are bound, to the double-stranded DNA; converting or deleting one or more nucleotides at the targeted site to one or more other nucleotides without cutting both strands of the double-stranded DNA at the targeted site; or inserting one or more nucleotides at the targeted site, the nucleic acid sequence recognition module being a CRISPR-Cas system in which both DNA-cleaving abilities of Cas are deactivated.SELECTED DRAWING: None

Described herein are CAS12A mutants from Lachnospiraceae bacterium and methods for use thereof. These mutants have enhanced DNA cleavage activities at non-canonical TTTT protospacer adjacent motifs (PAM) compared to the wild-type enzyme.

The subject invention provides a novel fluorescence-based, T5 exonuclease-amplified DNA cleavage assay for gyrase poisoning inhibitor discovery. According to the assay, multiple gyrase molecules can simultaneously bind to a plasmid DNA molecule to form multiple gyrase-DNA cleavage complexes on the same plasmid. These gyrase-DNA cleavage complexes, greatly stabilized by a gyrase poisoning inhibitor, and can be trapped by a detergent such as sarkosyl. Digestion of gyrase by proteinase K results in the production of small DNA fragments, which can be digested by T5 exonuclease. This fluorescence-based DNA cleavage HTS assay is also suitable for screening large compound libraries to identify inhibitors against DNA topoisomerases, including human DNA topoisomerase IIα.

The invention discloses an ICD inducer Ru (II) complex with photodynamic DNA breaking capacity and a preparation method and application thereof, and relates to the field of antitumor drugs, the structural formula of the Ru (II) complex is [Ru (dip) 2 (L)] (PF6) 2, and the Ru (II) complex can enter cell nucleus to break DNA and activate pyroptosis and cGAS-STING pathway mediated immunogenic death when applied to photodynamic therapy, so that the antitumor immune effect of a body is improved. In an in-vivo experiment, the vaccine group has a good effect on immune cells of a tumor microenvironment in a mouse, and the three-needle vaccine has a better anti-tumor immune effect; hamp, Hamp; the E staining result shows that the vaccine has no obvious pathological injury on the main organs of the mouse, and the vaccine is proved to have better biological safety.