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Composition and methods for regulated expression of a guide RNA/cas endonuclease complex

An endonuclease, complex technology, applied in the field of changing the genome of plant cells

Inactive Publication Date: 2017-11-28
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although several methods have been developed to target specific sites for modification in the genome of plants, there remains a need to prevent stable integration of recombinant expression cassettes and / or to provide regulated expression systems

Method used

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  • Composition and methods for regulated expression of a guide RNA/cas endonuclease complex
  • Composition and methods for regulated expression of a guide RNA/cas endonuclease complex
  • Composition and methods for regulated expression of a guide RNA/cas endonuclease complex

Examples

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example 1

[0254] by delivering a Cas9 endonuclease expression cassette and a single guide RNA (either as an RNA molecule or as a DNA expression cassette) Modification of target DNA sequences in plant cell genomes

[0255] The Cas9 gene (SEQ ID NO: 1) from Streptococcus pyogenes M1GAS (SF370) was maize codon-optimized according to standard techniques known in the art and introduced into the potato ST-LS1 intron (SEQ ID NO: 2 ) in order to eliminate the expression of the gene in Escherichia coli and Agrobacterium. To facilitate the nuclear localization of Cas9 protein in maize cells, the simian virus 40 (SV40) monotype N-terminal nuclear localization signal (MAPKKKRKV, SEQ ID NO: 3) and the Agrobacterium tumefaciens bitype VirD2 T-DNA boundary A nuclease carboxy-terminal nuclear localization signal (KRPRDRHDGELGGRKRAR, SEQ ID NO: 4) was incorporated into the amino- and carboxy-terminals of the Cas9 open reading frame, respectively. The maize optimized Cas9 gene was operatively linked...

example 2

[0265] Transient delivery of guide RNA in plant cells containing a Cas9 expression cassette stably integrated in their genome

[0266] To overcome the aforementioned issues, and further evaluate the feasibility of transient sgRNA delivery, maize lines containing a pre-integrated copy of Cas9 were generated.

[0267] Agrobacterium vectors containing Cas9 endonuclease expression cassettes driven by inducible or constitutive promoters

[0268] Two Agrobacterium vectors ( figure 1 and figure 2 ) into maize to create a pre-integrated genomic copy of SpyCas9. These Agrobacterium T-DNAs also contained the embryonic END2 promoter regulating the expression of the blue fluorescent gene (Am-Cyan) as a visible marker, and an interrupted copy of the Ds-Red gene under the maize histone 2B promoter. A portion of the Ds-Red sequence is replicated in direct orientation (369 bp fragment) and consists of two segments of the Ds-Red (RF-FP) gene consisting of a 343-bp sequence containing a ...

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Abstract

Compositions and methods are provided for regulated expression of a guide RNA / Cas endonuclease system in a plant cell, plant and seed. Compositions and methods are also provided for genome modification of a target sequence in the genome of a plant or plant cell. The methods and compositions employ a regulated guide RNA / Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed.

Description

[0001] This application claims the benefit of US Provisional Application No. 62 / 120421, filed February 25, 2015, which is hereby incorporated by reference in its entirety. technical field [0002] The present disclosure relates to the field of plant molecular biology, and in particular to methods for altering the genome of plant cells. [0003] References to Sequence Listings Submitted Electronically [0004] The official copy of this Sequence Listing is submitted electronically via EFS-Web as a Sequence Listing in ASCII format under the file name "20160129_BB2393PCT_ST25_SeqLst.txt", created on January 29, 2016, and is 76 kilobytes in size and is identical to this The instruction manual is submitted at the same time. The Sequence Listing contained in this file in ASCII format is part of the specification and is hereby incorporated by reference in its entirety. Background technique [0005] Recombinant DNA technology enables the insertion of DNA sequences into the genome ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8213C12N15/8237C12N9/22C12N15/8212
Inventor A.M.斯甘S.斯维塔舍夫
Owner PIONEER HI BRED INT INC