Cpf1 based transcription regulation systems in plants

A plant and transcription factor technology, applied in the direction of plant regeneration, plant genetic improvement, botany equipment and methods, etc.

Pending Publication Date: 2021-01-08
KWS SAAT SE & CO KGAA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is not clear from the prior art whether simply replacing the repressive domain with the activating domain in a Cpf1-based system would lead to activation of endogenous gene expression
In contrast, the prior art suggests that extensive modification and experimentation are required to provide Cpf1-based transcriptional activators that can be used in plant cells

Method used

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  • Cpf1 based transcription regulation systems in plants
  • Cpf1 based transcription regulation systems in plants
  • Cpf1 based transcription regulation systems in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0383] Example 1: TAL Transcription Factors for Transient Expression of Endogenous Morphogenetic Genes in Maize (Zm)

[0384] In one embodiment, commercially designed and constructed TAL transcription factors are used to temporarily enhance the expression of BBM and WUS. The TAL transcription factor is designed to combine with the regulatory region of about 24 bp shown in SEQ ID NO:95, 109-147, 270-272 of BBM and / or the regulatory region of about 18 bp shown in SEQ ID NO:96, 148-190 of WUS (See Figures 3A and 3B). The TAL transcription factor recognition domain of BBM comprises the sequence shown in SEQ ID NO: 13-51, and / or the TAL transcription factor recognition domain of WUS comprises the sequence shown in SEQ ID NO: 52-94.

[0385] TAL effector sequences can be designed and cloned, and the activation domain of herpes simplex virus (VP16 or tetrameric VP64) can be added to the construct in a fusion protein-like manner.

[0386]Transient induction of expression was first t...

Embodiment 2

[0389] Example 2: Fusion protein between non-functional CRISPR-nuclease and activation domain for transient expression of endogenous morphogenetic genes in maize

[0390] Similar to Example 1, constructs were designed for transient delivery, in this case expressing dCas9 (PAM variant available) or dCpf1 (PAM variant available) as a protein with an activation domain (e.g., VP16 or VP64). fusion protein. Potential target sites / regulatory regions include: the Cas9 target sequence shown in SEQ ID Nos: 97-99 of ZmBBM; the Cpf1 target sequence shown in SEQ ID Nos: 100-102 of ZmBBM; the SEQ ID NOs of ZmWUS2: 103- Cas9 target sequence shown in 105; Cpf1 target sequence shown in SEQ ID Nos: 106-108 of ZmWUS2.

[0391] Based on the regulatory regions of CRISPR / dCas9 and CRISPR / dCpf1 described above, a CRISPR-based transcription factor system having a recognition domain comprising the sequence shown in SEQ ID NO: 1-12 can be designed and commercially obtained.

[0392] Transient induct...

Embodiment 3

[0394] Example 3: Replacement of Activation Domains to Optimize Expression of Morphogenetic Genes

[0395] This example aims to test the behavior of different aforementioned activation domains in a systematic manner. This was sufficient to assess their effect on the expression levels of ZmWUS and ZmBBM. As noted above, different STFs for a specific target gene of interest may contain different activation and recognition domains and other elements. Therefore, it is very suitable for designing different STFs for one target and the same target, so as to finally define the optimal STF for regulating the gene of interest.

[0396] The native activation domains of the TAL effector genes of Xanthobacterium oryzae are the most obvious activation domains to use with TAL transcription factors, and also represent an activation domain that can be used alone or in combination according to various aspects of the invention but has also been used in Other set activation domains. They belon...

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PUM

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Abstract

The present invention relates to the targeted regulation of gene expression and more specifically to synthetic transcription factors (STFs) comprising at least one highly target specific engineered recognition domain based on a CRISPR / Cpf1 system and further comprising at least one activation or silencing domain to modulate the expression of a gene of interest, preferably to modulate the transcription of a morphogenic gene of a eukaryote, in particular a plant. Further disclosed are methods using the STFs to enhance transformation frequencies, to optimize successful genome editing approaches,to provide haploid or double haploid organisms, and / or to provide compositions suitable for general transformation, but also for breeding purposes.

Description

technical field [0001] The present invention relates to targeted regulation of gene expression, and more specifically to synthetic transcription factors (STFs) comprising at least one highly target-specific engineered recognition domain based on the CRISPR / Cpf1 system, and at least one activation or silencing domain To regulate the expression of the target gene, it is preferable to regulate the transcription of morphogenetic genes of eukaryotes, especially plants. Also disclosed are methods of using STFs to increase transformation frequency, optimize successful genome editing methods, provide haploid or double haploid organisms, and / or provide compositions suitable for general transformation but also for breeding purposes. These methods and uses rely on the synergistic effect of STFs comprising gene expression regulatory domains (e.g., activation domains or silencing domains) such that cells can be reprogrammed and induced to divide and / or regenerate while transforming said ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/63A01H4/00A01H1/08
CPCA01H1/08A01H4/00C12N15/63C12N15/8201C12N15/8207C12N15/8213C12N15/8216C12N15/8217C12N2310/20
Inventor M·拉布斯A·赫梅尔梅钰
Owner KWS SAAT SE & CO KGAA
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