US 12,098,374 B2 · Optimized plant CRISPR/CPF1 systems

Abstract

The present invention relates to optimized systems for the genome editing of eukaryotic cells, preferably plant cells. Also provided is a plant delivery system comprising at least one Cpf1 enzyme, or a plant optimized construct encoding the same, to be synergistically combined with a Cpf1 guide RNA system. Further provided is a Cpf1 guide RNA system being flanked by a Hammerhead ribozyme sequence at the 5′ end and by a plant-derived Hepatitis Delta Vims (HDV)-like ribozyme sequence at the 3′ end, or being embedded within a coding or non-coding region, of a sequence encoding a frame sequence. Novel plant-derived HDV-like ribozyme sequences are also provided. Further provided are methods for improved genome editing, and the use of the various systems provided herein to obtain transformed plants, plant cells, tissues, organs, or a progeny thereof, or a plant material, modified in a targeted way even at difficult to access target sites.

Claims44

Dependent claims

A plant delivery system, the delivery system comprising

(a) at least one Cpf1 enzyme or an active fragment thereof, or a nucleic acid sequence encoding the same, wherein the at least one nucleic acid sequence encoding the Cpf1 enzyme or an active fragment thereof is codon-optimized for the expression in the plant or part of the plant;
and
(b) at least one Cpf1 guide RNA system, or a nucleic acid sequence encoding the same, the at least one Cpf1 guide RNA system comprising at least one Cpf1 guide RNA specific for a genomic target sequence of interest in a plant or part of a plant;
wherein the at least one Cpf1 guide RNA, or the nucleic acid sequence encoding the same, is
(i) flanked by a Hammerhead ribozyme sequence at the 5′-end and by a plant-derived Hepatitis Delta Virus (HDV)-like ribozyme sequence at the 3′-end, wherein the plant-derived HDV-like ribozyme sequence, or a sequence encoding the same, is selected from any one of SEQ ID NOs:
19, 21, 25, and sequences having at least 95% identity over the whole length of any one of SEQ ID NOs:
19, 21, or 25;
and is
(ii) embedded within a 3′ untranslated region (UTR) of a sequence encoding a frame sequence.

6 more elements

A method for modifying a genomic target sequence of interest in a plant or part of a plant, wherein the method comprises the steps of:

(a) providing at least one Cpf1 enzyme or an active fragment thereof, or a nucleic acid sequence encoding the same, wherein the at least one nucleic acid sequence encoding the Cpf1 enzyme or an active fragment thereof is codon-optimized for the expression in the plant or part of the plant;
and
(b) providing at least one Cpf1 guide RNA system, or a nucleic acid sequence encoding the same, the at least one Cpf1 guide RNA system comprising at least one Cpf1 guide RNA specific for a genomic target sequence of interest in the plant or part of the plant;
wherein
(i) the at least one Cpf1 guide RNA, or the nucleic acid sequence encoding the same, is flanked by a Hammerhead ribozyme sequence at the 5′ and a plant-derived Hepatitis Delta Virus (HDV)-like ribozyme sequence at the 3′-end, wherein the plant-derived HDV-like ribozyme sequence, or a sequence encoding the same, is selected from any one of SEQ ID NOs:
19, 21, and 25, and sequences having at least 95% identity over the whole length of any one of SEQ ID NOs:
19, 21, or 25;
and
(ii) the at least one Cpf1 guide RNA, or the nucleic acid sequence encoding the same, is embedded within a 3′ untranslated region (UTR) of a sequence encoding a frame sequence;
(c) optionally:
providing at least one repair template nucleic acid sequence, wherein the at least one repair template nucleic acid sequence is flanked by one or more homology sequence(s) complementary to one or both adjacent region(s) of the genomic sequence of interest in the plant or part of the plant;
(d) introducing the at least one Cpf1 enzyme or an active fragment thereof, or a nucleic acid sequence encoding the same from step (a);
and introducing the at least one Cpf1 guide RNA system, or a nucleic acid sequence encoding the same from step (b) and optionally:
introducing the at least one repair template nucleic acid sequence from step (c) into the plant or part of the plant;
and
(e) obtaining a plant or part of a plant, or a progeny thereof, comprising a modification in the genomic target sequence of interest.

13 more elements

Dependencies are inferred from the claim text.

Optimized plant CRISPR/CPF1 systems

Life of this patent

Optimized plant CRISPR/CPF1 systems

Life of this patent

Abstract

Claims44