Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genomic engineering, epigenomic engineering, genome targeting, and genome editing.

A method for identifying the location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and optionally reacting a second portion of the sample with a dioxygenase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase that is more efficient at converting methylcytosine to carboxymethylcytosine is also provided.

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

Provided herein are, inter alia, compositions and methods for demethylating and methylating a target DNA sequences in a mammalian cell. The compositions and methods are, useful for activity modulation of a targeted gene, or to create a gene regulatory network.

In order to provide: a method for producing an HSL protein with increased catalytic activity to oxidize a 4-HPPD inhibitor in a 2-oxoglutarate-dependent manner; and a method for producing a plant with increased resistance to a 4-HPPD inhibitor using the method for producing the HSL protein, it has been revealed that, by mutating position 140 to a basic amino acid in an HSL protein, the catalytic activity of the protein to oxidize a 4-HPPD inhibitor in a 2-oxoglutarate-dependent manner can be increased, and an activity of the protein to decompose the inhibitor can be increased.

Many studies have shown that CRISPR-Cas nucleases can tolerate up to five mismatches and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using RNA-guided Foki Nucleases (RFNs), e.g., Fokl-Cas9 or Foki-dCas9-based fusion proteins.

Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genomic engineering, epigenomic engineering, genome targeting, and genome editing.

Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map .sup.5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map .sup.5hmC in a DNA.

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems.

A protein having an L-proline efflux function and the use thereof are provided. A method for producing L-proline or hydroxyproline by means of using a protein ThrE is used for producing L-proline by means of enhancing the activity of a polypeptide, having an L-proline efflux function, in an L-proline-producing strain. Alternatively, the method is used for producing hydroxyproline by means of weakening the activity of a polypeptide, having an L-proline efflux function, in L-proline-producing host cells and enhancing the activity of a proline hydroxylase.