Pioneer Hi-Bred International, Inc.

Vilnius University

Virginijus Siksnys

Tautvydas Karvelis

Joshua K Young

Zhenglin Hou

Gintautas Tamulaitis

Giedrius Gasiunas

Monika Glemzaite

Arvydas Lubys

Anja Smith

Andrew Mark Cigan

Lolita Zaliauskiene

Stephane Deschamps

Migle Kazlauskiene

Giedrius Gasiūnas

Miglé Kazlauskiene

Matthias Bochtler

Thomas Fricke

Monika Gasiuniene

Mindaugas Zaremba

Georgij Kostiuk

Compositions and methods are provided for genome modification of a target sequence in the genome of a cell, using a novel Cas endonuclease. The methods and compositions employ a guide polynucleotide/endonuclease system to provide an effective system for modifying or altering target sequences within the genome of a cell or organism. Also provided are novel effectors and endonuclease systems and elements comprising such systems, such as guide polynucleotide/endonuclease systems comprising an endonuclease. Compositions and methods are also provided for guide polynucleotide/endonuclease systems comprising at least one endonuclease, optionally covalently or non-covalently linked to, or assembled with, at least one additional protein subunit, and for compositions and methods for direct delivery of endonucleases as ribonucleotide proteins.

CRISPR-Cas systems for genome editing

CRISPR-cas systems for genome editing

Compositions and methods are provided for novel Cas9 orthologs, including, but not limiting to, novel guide polynucleotide/Cas9 endonucleases complexes, single or dual guide RNAs, guide RNA elements, and Cas9 endonucleases. The present disclosure also describes methods for creating a double strand break in a target polynucleotide, methods for genome modification of a target sequence under various in vivo and in vitro conditions, in the genome of a cell, for gene editing, and for inserting a polynucleotide of interest into the genome of a cell. Also provided are nucleic acid constructs and cells having a modified target site or altered polynucleotide of interest produced by the methods described herein.

Cas9 orthologs

Methods and compositions are provided for the identification, detection, characterization, and/or utilization of double strand breaks in a target polynucleotide; the identification, detection, characterization, and/or utilization of cutting sites for double-strand-break-inducing agents; and the identification, detection, characterization, and/or utilization of double-strand-break-inducing agents.

Methods for the identification and characterization of double-strand break sites and compositions and uses thereof

Isolation or in vitro assembly of the Cas9-crRNA complex of theCRISPR3/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 in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences 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 and using a cassette containing a single repeat-spacer-repeat unit.

RNA-directed DNA cleavage by the Cas9-crRNA complex

Compositions and methods are provided for rapid characterization of Cas endonuclease systems and the elements comprising such systems, including, but not limiting to, rapid characterization of PAM sequences, guide RNA elements and Cas endonucleases. Type II Cas9 endonuclease systems originating fromMlc3,DSM 15814,SL4,JCM 14932,sp. SCADC,DSM 20210,NX02,DSM 16811,are described herein. The present disclosure also describes methods for genome modification of a target sequence in the genome of a cell, for gene editing, and for inserting a polynucleotide of interest into the genome of a cell.

Rapid characterization of Cas endonuclease systems, PAM sequences and guide RNA elements

Methods and compositions using a CRISPR-Cas Type IIIA resulting in RNA gene knockdown and knockout in an animal.

Targeted RNA knockdown and knockout by type III-A Csm complexes

CRISPR-CAS systems for genome editing

Isolation or in vitro assembly of the Cas9-crRNA complex of theCRISPR3/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.

A Type III-A CRISPR-Cas (StCsm) complex ofcomprising crRNA, Csm4, and Csm3 and use for cleavage of RNA bearing a nucleotide sequence complementary to the crRNA, in vitro or in vivo. Methods for site-specific cleavage/shredding of a target RNA molecule using an RNA-guided RNA endonuclease comprising a minimal complex of crRNA, Csm4, and Csm3, and methods of RNA knock-down and RNA knock-out are disclosed.

Growing community of inventors

Vilnius, Lithuania

Virginijus Siksnys