1121 results about "DNA Endonuclease" patented technology

Disclosed is a nuclease resistant nucleotide compound capable of hybridizing with a complementary RNA in a manner which inhibits the function thereof, which modified nucleotide compound includes at least one component selected from the group consisting of MN₃M, B(N)_xM and M(N)_xB wherein N is a phosphodiester-linked modified 2′-deoxynucleoside moiety; M is a moiety that confers endonuclease resistance on said component and that contains at least one modified or unmodified nucleic acid base; B is a moiety that confers exonuclease resistance to the terminus to which it is attached; and x is an integer of at least 2.

Provided herein are methods for inactivating a target polynucleotide. The methods use a psiRNA having a 5′ region and a 3′ region. The 5′ region includes, but is not limited to, 5 to 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer. The 3′ region is substantially complementary to a portion of the target polynucleotide. The methods may be practiced in a prokaryotic microbe or in vitro. Also provided are polypeptides that have endonuclease activity in the presence of a psiRNA and a target polynucleotide, and methods for using the polypeptides.

The present invention provides methods for modifying chromosomal sequences. In particular, methods are provided for using RNA-guided endonucleases or modified RNA-guided endonucleases to modify targeted chromosomal sequences.

The invention provides a method for constructing a high resolution physical map of a polynucleotide. In accordance with the invention, nucleotide sequences are determined at the ends of restriction fragments produced by a plurality of digestions with a plurality of combinations of restriction endonucleases so that a pair of nucleotide sequences is obtained for each restriction fragment. A physical map of the polynucleotide is constructed by ordering the pairs of sequences by matching the identical sequences among the pairs.

Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific recombination, using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.

The present invention provides a method for identifying one or more low abundance sequences differing by one or more single-base changes, insertions, or deletions, from a high abundance sequence in a plurality of target nucleotide sequences. The high abundance wild-type sequence is selectively removed using high fidelity polymerase chain reaction analog conversion, facilitated by optimal buffer conditions, to create a restriction endonuclease site in the high abundance wild-type gene, but not in the low abundance mutant gene. This allows for digestion of the high abundance DNA. Subsequently the low abundant mutant DNA is amplified and detected by the ligase detection reaction assay. The present invention also relates to a kit for carrying out this procedure.

The present invention generally relates to the determination of the state of one or more locations within a nucleic acid and, in particular, to the determination of the methylation state of one or more methylation sites within a nucleic acid such as DNA. In one aspect of the invention, a nucleic acid, such as DNA, that is suspected of being methylated is exposed to a nucleic acid probe able to hybridize the nucleic acid at or near the methylation site. After hybridization, the nucleic acid-probe hybrid is exposed to a methylation-sensitive restriction endonuclease able to bind at or near the methylation site. The restriction endonuclease is not able to cleave the nucleic acid-probe hybrid if the DNA is methylated at the methylation site, but is able to cleave the nucleic acid-probe hybrid if the nucleic acid is not methylated at the methylation site. Determination of the cleavage state of the probe can thus be used to determine the state of the methylation site.

The present invention provides novel cleavage agents and polymerases for the cleavage and modification of nucleic acid. The cleavage agents and polymerases find use, for example, for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. In some embodiments, the 5′ nuclease activity of a variety of enzymes is used to cleave a target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

The present invention relates to methods of developing genetically engineered, preferably non-alloreactive T-cells for immunotherapy. This method involves the use of RNA-guided endonucleases, in particular Cas9/CRISPR system, to specifically target a selection of key genes in T-cells. The engineered T-cells are also intended to express chimeric antigen receptors (CAR) to redirect their immune activity towards malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer and viral infections.

A method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) in the proviral DNA, and inactivating the proviral DNA. A composition for use in inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus including isolated nucleic acid sequences comprising a CRISPR-associated endonuclease and a guide RNA, wherein the guide RNA is complementary to a target sequence in a human immunodeficiency virus.

The present invention relates to methods of developing genetically engineered, preferably non-alloreactive T-cells for immunotherapy. This method involves the use of RNA-guided endonucleases, in particular Cas9/CRISPR system, to specifically target a selection of key genes in T-cells. The engineered T-cells are also intended to express chimeric antigen receptors (CAR) to redirect their immune activity towards malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer and viral infections.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide mRNA-sensing gRNAs that modulate the activity of RNA-programmable endonucleases based on the presence or absence of a target mRNA.

The invention provides a construction method and application of a genome-wide methylation high-throughput sequencing library. The construction method of the genome-wide methylation high-throughput sequencing library comprises steps of: conducting digestion on a genome DNA with Msp I and a second restriction endonuclease; conducting end repair on DNA fragments; adding a basic group A on a 3'terminal of the DNA fragment subjected to end repair; connecting a DNA fragment with a cohesive end A to a methylation joint; conducting fragment selection on the connect products with the methylation joint, in order to obtain a target fragment; subjecting the target fragment to a bisulfite treatment, in order to convert unmethylated cytosine in the target fragment to uracil; subjecting the converted target fragment to PCR amplification; and separating and purifying the amplification products, wherein the amplification products form the genome-wide methylation high-throughput sequencing library. The construction method and application of the genome-wide methylation high-throughput sequencing library provided by the invention can conveniently and effectively construct the genome-wide methylation high-throughput sequencing library of the genome DNA sample.