Pioneer Hi-Bred International, Inc.

Vilnius University

Tautvydas Karvelis

Virginijus Siksnys

Joshua K Young

Zhenglin Hou

Giedrius Gasiunas

Anja Smith

Arvydas Lubys

Monika Glemzaite

Andrew Mark Cigan

Lolita Zaliauskiene

Monika Gasiuniene

Giedrius Gasiūnas

Mindaugas Zaremba

Paulius Toliusis

Evelina Zagorskaite

Giedrius Sasnauskas

Arunas Silanskas

Tomas Sinkunas

Algirdas Grybauskas

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

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

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.

Growing community of inventors

Vilnius, Lithuania

Tautvydas Karvelis