CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

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Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

Methods and devices are provided for providing surgical access into a body cavity. In one embodiment, a surgical access device is provided that includes a housing coupled to a retractor. The housing can be have one or more movable sealing ports for receiving surgical instruments. Each movable sealing port can include one or more sealing elements therein for sealing the port and/or forming a seal around a surgical instrument disposed therethrough. Each movable sealing port can be rotatable relative to the housing and each sealing element can be rotatable relative to the housing along a predetermined orbital path.

Methods and devices for providing access into a body cavity

Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.

Methods and apparatuses for analyzing polynucleotide sequences

The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.

Short cycle methods for sequencing polynucleotides

The invention provides methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension. Some applications of the invention utilize total internal reflection fluorescence microscopy to image polynucleotide molecules at single molecule resolution.

Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.

/pdf/improving-crispr-cas-specificity-with-chemical-modifications-574ycolnh5.pdf

Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

To probe the characteristics of a target nucleic acid molecule or a population of target nucleic acid molecules, a local cross-sectional area, local charge, or local chemistry of a molecule is modified. The modified nucleic acid is contacted with a substrate that includes a detector that is responsive to the modification in the local cross-sectional area, local charge, or local chemistry of the nucleic acid molecule. The modified nucleic acid molecule traverses a defined and preferably molecular dimensioned volume on the substrate so that nucleotides of the modified nucleic acid molecule interact with the detector in sequential order, whereby data correlating with the cross-sectional area, local charge, or local chemistry of the nucleic acid molecule are obtained. The nucleic acid molecule may be modified in a number of locations along the molecule's length. Because the individual nucleotides of the nucleic acid molecule interact with the detector in sequential order, information regarding the location and composition of a plurality of modified sites along a single molecule can be obtained.

Methods for characterization of nucleic acid molecules

An improved method for the chemical synthesis of RNA was developed utilizing a streamlined method for the preparation of phosphoramidite monomers and a single-step deprotection of the resulting oli...

Streamlined Process for the Chemical Synthesis of RNA Using 2′-O-Thionocarbamate-Protected Nucleoside Phosphoramidites in the Solid Phase

The present invention provides a system for generating nucleic acid molecules having a reduced ability to hybridize to form intermolecular and intramolecular base pairs between regions of substantial complementarity as compared with nucleic acid molecules having the same nucleotide sequence containing naturally-occurring nucleotides. Furthermore, nucleic acid molecules of the present invention are characterized by the ability to form intermolecular base pairs with other nucleic acid molecules of choice.

Method of producing nucleic acid molecules with reduced secondary structure

Methods and reagents are disclosed which provide for more sensitive, more accurate and higher through-put analyses of target nucleic acid sequences. The methods and reagents of the present invention may be generically applied to generally any target nucleic acid sequence and do not require a priori information about the presence, location or identity of mutations in the target nucleic acid sequence. The reagents of the invention are mixtures of oligonucleotide precursors having a high level of coverage and mass number complexity, and also having tags analyzable by mass spectrometry which are covalently linked to the precursors through cleavable bonds. A method is also disclosed for analyzing a target nucleic acid sequence employing the mixtures of oligonucleotide precursors having tags analyzable by mass spectrometry covalently linked to the oligonucleotide precursors through cleavable bonds, and chemical or enzymatic assays to alter the mass of the oligonucleotide precursors prior to mass spectral analysis. The enzymatic assay may be a polymerase extension assay or a ligation-based assay. The kits for carrying out the methods of the invention are also disclosed.

Jeffrey R. Sampson

Papers

Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

Methods for characterization of nucleic acid molecules

Streamlined Process for the Chemical Synthesis of RNA Using 2′-O-Thionocarbamate-Protected Nucleoside Phosphoramidites in the Solid Phase

Method of producing nucleic acid molecules with reduced secondary structure

Method and reagents for analyzing the nucleotide sequence of nucleic acids