US 2021/0071189 A1 · CPF1 BASED TRANSCRIPTION REGULATION SYSTEMS IN PLANTS

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.

Claims58

Dependent claims

A synthetic transcription factor, or a nucleotide sequence encoding the same, comprising at least one recognition domain and at least one activation domain, wherein the synthetic transcription factor is configured to modulate the expression of a morphogenic gene in a cellular system.

A method for increasing the transformation efficiency in a cellular system, wherein the method comprises the steps of:

(a) providing a cellular system;
(b) introducing into the cellular system at least one synthetic transcription factor, or a nucleotide sequence encoding the same;
and
(c) introducing into the cellular system at least one nucleotide sequence of interest;
(d) optionally:
culturing the cellular system under conditions to obtain a transformed progeny of the cellular system;
wherein the at least one synthetic transcription factor, or the nucleotide sequence encoding the same, comprises at least one recognition domain and at least one activation domain, wherein the synthetic transcription factor is configured to modulate the expression, preferably the transcription, of at least one morphogenic gene in the cellular system;
and
wherein the at least one synthetic transcription factor, or the nucleotide sequence encoding the same, is introduced in parallel to, or sequentially with the introduction of the at least one nucleotide sequence of interest.

6 more elements

A method for increasing the expression of at least one endogenous gene in a cellular system, wherein the method comprises the steps of:

(a) providing a cellular system;
(b) introducing into the cellular system at least one synthetic transcription factor, or a nucleotide sequence encoding the same;
wherein the at least one synthetic transcription factor, or the nucleotide sequence encoding the same, comprises at least one recognition domain and at least one activation domain, wherein the synthetic transcription factor is configured to increase the expression, preferably the transcription, of at least one endogenous gene in the cellular system.

A method of modifying the genetic material of a cellular system at a predetermined location, wherein the method comprises the following steps:

(a) providing a cellular system;
(b) introducing at least one synthetic transcription factor, or a sequence encoding the same, into the cellular system,
(c) further introducing into the cellular system
(i) at least one site-specific nuclease, or a sequence encoding the same, wherein the site-specific nuclease induces a double-strand break at the predetermined location;
(ii) optionally:
at least one nucleotide sequence of interest, preferably flanked by one or more homology sequence(s) complementary to one or more nucleotide sequence(s) adjacent to the predetermined location in the genetic material of the cellular system;
and;
(e) optionally:
determining the presence of the modification at the predetermined location in the genetic material of the cellular system;
and
(f) obtaining a cellular system comprising a modification at the predetermined location of the genetic material of the cellular system;
wherein the at least one synthetic transcription factor, or the nucleotide sequence encoding the same, comprises at least one recognition domain and at least one activation domain, wherein the at least one synthetic transcription factor is configured to modulate the expression, preferably the transcription, of at least one morphogenic gene in the cellular system;
and
wherein the at least one synthetic transcription factor, or the nucleotide sequence encoding the same, is introduced in parallel to, or sequentially with the introduction of the at least one site-specific nuclease, or the sequence encoding the same and the optional at least one nucleotide sequence of interest.

11 more elements

A method of producing a haploid or double haploid organism, wherein the method comprises the following steps:

(a) providing a haploid cellular system;
(b) introducing into the haploid cellular system at least one synthetic transcription factor, or a nucleotide sequence encoding the same;
(c) culturing the haploid cellular system under conditions to obtain at least one haploid or double haploid organism;
and
(d) optionally:
selecting the at least one haploid or double haploid organism obtained in step (c),
wherein the at least one synthetic transcription factor, or the nucleotide sequence encoding the same, comprises at least one recognition domain and at least one activation domain, wherein the at least one synthetic transcription factor is configured to modulate the expression, preferably the transcription, of at least one morphogenic gene in the haploid cellular system.

4 more elements

Dependencies are inferred from the claim text.

Cpf1 based transcription regulation systems in plants

Life of this patent

Cpf1 based transcription regulation systems in plants

Life of this patent

Abstract

Claims58