Keygene N.v.

Dow Agrosciences LLC

Sangamo Therapeutics, Inc.

Sangamo Biosciences, Inc.

Corteva Agriscience LLC

Paul Bundock

Fyodor Urnov

Manju Gupta

Lakshmi Sastry-Dent

Dmitry Y Guschin

Vipula Shukla

Michiel Theodoor Jan De Both

Jeroen Stuurman

René Cornelis Josephus Hogers

Michiel Jan De Both

Franck Lhuissier

Anita Ketelaars-Bonné

Frank Lhuissier

Franck Georges Paul Lhuissier

Anita Ketelaars-BonnÉ

Bernarda Gerharda Johanna Fierens-Onstenk

Ludvik Kevin Wachowski

The current invention relates to methods of targeted genetic alteration in plant cells, as well as to plant cells and plants thus obtained. In the method, a Cpf1 protein and a crRNA is employed to provide for targeted alteration, in particular increased biallelic alteration, of a DNA duplex with increased efficacy.

Method for targeted alteration of duplex DNA

The invention pertains to the targeted alteration of a duplex DNA in a cell, whereby two site-specific nucleases generate an indel, such that the open reading frame is not altered after the second indel. The invention further pertains to the use of such nucleases for the targeted alteration of an open reading frame in duplex DNA and a kit of parts for use in a method of the invention. Using the method of the invention, novel plants were obtained having an improved herbicide resistance. The invention therefore also concerns plants having improved herbicide resistance due to the expression of an altered ALS protein.

Balanced indels

The current invention relates to methods of targeted genetic alteration in cells, preferably plant cells, as well as to plant cells and plants thus obtained using at least a fusion protein comprising a site-specific nuclease domain and a deaminase domain, or a construct encoding the same. The method also provides for a composition and a kit comprising a combination of a first fusion protein comprising a cytosine deaminase domain and a second fusion protein comprising an adenine deaminase domain, preferably for use in the method of the invention. The method provides for targeted alteration of a DNA duplex in plant cells with increased efficacy.

Methods of targeted genetic alteration in plant cells

The current disclosure relates to the field of plants, in particular to the fields of plant breeding and plant genetics. More particular, the disclosure concerns inventive methodology that may be useful in improving plant properties. In particular the invention may be useful in removing linkage drag. Also provided are plant and plant parts obtained with the method disclosed herein.

Method for removing genetic linkage in a plant

Disclosed is a new method of providing plant cells with a targeted alteration in a DNA molecule. The method comprises contacting a population of plant cells comprising a DNA molecule, the DNA molecule having a target sequence, with an aqueous medium, wherein the aqueous medium comprises a CRISPR associated protein (CAS protein) or a CAS-like protein, and a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and wherein the aqueous medium comprises polyethylene glycol (PEG), but needs to be substantially free of glycerol.

Method for targeted DNA alteration in plant cells

Disclosed herein are methods and compositions for targeted modification of one or more endogenous plant malate dehydrogenase genes.

Targeted modification of malate dehydrogenase

Method for targeted alteration of a duplex acceptor DNA sequence in a plant cell protoplast, comprising combining the duplex acceptor DNA sequence with a donor mutagenic nucleobase, wherein the duplex acceptor DNA sequence contains a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence and wherein the donor mutagenic nucleobase comprises at least one mismatch with respect to the duplex acceptor DNA sequence to be altered, preferably with respect to the first DNA sequence, wherein the method further comprises a step of introducing the donor mutagenic nucleobase into the cell protoplasts using polyethylene glycol (PEG) mediated transformation and the use of PEG protoplast transformation for enhancing the rate of targeted mutagenesis.

Mutagenesis method using polyethylene glycol mediated introduction of mutagenic nucleobases into plant protoplasts

The present invention relates to a new method for improving glyphosate resistance of a plant. The method encompasses providing one or more specific mutations in a specific nucleotide sequence in said plant. In comparison to a plant not manipulated according to the method, the plant obtained by the method displays (improved) glyphosate resistance. Also provided are a (transgenic) plant, including a seed thereof, and a plant product that can be obtained by the method according to the invention.

Glyphosate resistance enhancement

The current invention relates to a method for targeted alteration of acceptor DNA, for example duplex acceptor DNA. The method comprises the use of an oligonucleotide having at least one mismatch relative to the targeted (duplex) acceptor DNA. The mismatch is located at specific positions within said oligonucleotide. Also provided is a kit that comprises instructions for performing the method according to the inventions, and in a preferred embodiment, comprises an oligonucleotide suitable for use in the method.

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Wageningen, Netherlands

Paul Bundock