Guide polynucleotide multiplexing
AssigneePioneer Hi-Bred International, Inc.
InventorsWILLIAM JAMES GORDON-KAMM, JOSHUA K. YOUNG
Life of this patent
Estimating the term…
Abstract
The present disclosure relates to methods and compositions for expressing multiple guide polynucleotides from one or more transcripts. Compositions and methods for delivering a plurality of guide polynucleotides to target multiple independent sites in a cell's genome are also provided.
Claims130
A method of delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the method comprising:
- providing a polynucleotide molecule to the plant cell, the polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are separated by a double-stranded RNA-specific RNase III recognition sequence;
- and
- expressing a eukaryotic RNase III in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide, wherein the RNA molecule having the RNase III recognition sequence comprises at least one stem loop.
A composition for delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the composition comprising:
- (a) a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are separated by a double-stranded RNA-specific RNase III recognition sequence;
- and
- (b) a heterologous eukaryotic RNase III that cleaves an RNA molecule transcribed from the polynucleotide molecule, wherein the RNA molecule having the RNase III recognition sequence comprises at least one stem loop.
A method for editing a plant genome, the method comprising:
- providing a plant cell with:
- (a) a Cas endonuclease;
- and
- (b) a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are separated by a double-stranded RNA-specific RNase III recognition sequence;
- expressing a eukaryotic RNase III in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide, wherein the RNA molecule having the RNase III recognition sequence comprises at least one stem loop;
- introducing a first site-specific modification in a first target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the first guide polynucleotide;
- and
- introducing a second site-specific modification in a second target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the second guide polynucleotide.
A method of delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the method comprising:
- providing a polynucleotide molecule to the plant cell, the polynucleotide molecule comprising an RNase Z recognition sequence, a self-cleaving ribozyme-encoding nucleotide sequence, and at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are flanked by the self-cleaving ribozyme-encoding nucleotide sequence at a 3′ end of the polynucleotide molecule and at least one of the two guide polynucleotide sequences is flanked by the RNase Z recognition sequence at a 5′ end;
- and
- expressing the polynucleotide molecule in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide.
A composition for delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the composition comprising a polynucleotide molecule comprising an RNase Z recognition sequence, a self-cleaving ribozyme-encoding nucleotide sequence, and at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are flanked by the self-cleaving ribozyme-encoding nucleotide sequence at a 3′ end of the polynucleotide molecule and at least one of the two guide polynucleotides is flanked by the RNase recognition sequence at a 5′ end.
A method for editing a plant genome, the method comprising:
- providing a plant cell with:
- (a) a Cas endonuclease;
- and
- (b) a polynucleotide molecule comprising an RNase Z recognition sequence, a self-cleaving ribozyme-encoding nucleotide sequence, and at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are flanked by the self-cleaving ribozyme-encoding nucleotide sequence at a 3′ end of the polynucleotide molecule and at least one of the two guide polynucleotide sequences is flanked by the RNase Z recognition sequence at a 5′ end,
- expressing the polynucleotide molecule in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide;
- introducing a first site-specific modification in a first target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the first guide polynucleotide;
- and
- introducing a second site-specific modification in a second target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the second guide polynucleotide.
A method of delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the method comprising:
- providing a polynucleotide molecule to the plant cell, the polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are separated by a double-stranded RNA-specific protein recognition sequence;
- and
- expressing a double-stranded RNA-specific protein comprising a ribonuclease domain in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide, wherein the RNA molecule having the double-stranded RNA-specific protein recognition sequence comprises at least one stem loop.
A composition for delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the composition comprising:
- (c) a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are separated by a double-stranded RNA-specific protein recognition sequence;
- and
- (d) a double-stranded RNA-specific protein comprising a ribonuclease domain that cleaves an RNA molecule transcribed from the polynucleotide molecule, wherein the RNA molecule having the double-stranded RNA-specific protein recognition sequence comprises at least one stem loop.
A method for editing a plant genome, the method comprising:
- providing a plant cell with:
- (c) a Cas endonuclease;
- and
- (d) a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein the at least two guide polynucleotide sequences are separated by a double-stranded RNA-specific protein recognition sequence;
- expressing a double-stranded RNA-specific protein comprising a ribonuclease domain in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide, wherein the RNA molecule having the double-stranded RNA-specific protein recognition sequence comprises at least one stem loop;
- introducing a first site-specific modification in a first target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the first guide polynucleotide;
- and
- introducing a second site-specific modification in a second target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the second guide polynucleotide.
A method of delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the method comprising:
- providing a polynucleotide molecule to the plant cell, the polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein each of the at least two guide polynucleotide sequences is flanked by an RNase Z recognition sequence;
- and
- expressing the polynucleotide molecule in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide.
A composition for delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the composition comprising a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein each of the at least two guide polynucleotide sequences is flanked by an RNase recognition sequence.
A method for editing a plant genome, the method comprising:
- providing a plant cell with:
- (c) a Cas endonuclease;
- and
- (d) a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein each of the at least two guide polynucleotide sequences is flanked by an RNase Z recognition sequence,
- expressing the polynucleotide molecule in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide;
- introducing a first site-specific modification in a first target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the first guide polynucleotide;
- and
- introducing a second site-specific modification in a second target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the second guide polynucleotide.
A method of delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the method comprising:
- providing a polynucleotide molecule to the plant cell, the polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein each of the at least two guide polynucleotide sequences is flanked by a self-cleaving ribozyme-encoding nucleotide sequence;
- and
- expressing the polynucleotide molecule in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide.
A composition for delivering a plurality of guide polynucleotides for multiplexed genome editing of multiple genomic targets in a plant cell, the composition comprising a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein each of the at least two guide polynucleotide sequences is flanked by a self-cleaving ribozyme-encoding nucleotide sequence.
A method for editing a plant genome, the method comprising:
- providing a plant cell with:
- (e) a Cas endonuclease;
- and
- (f) a polynucleotide molecule comprising at least two guide polynucleotide sequences targeting at least two distinct genomic target sites in the plant cell, wherein each of the at least two guide polynucleotide sequences is flanked by a self-cleaving ribozyme-encoding nucleotide sequence,
- expressing the polynucleotide molecule in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule, thereby delivering the at least two guide polynucleotide sequences as a first guide polynucleotide and a second guide polynucleotide;
- introducing a first site-specific modification in a first target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the first guide polynucleotide;
- and
- introducing a second site-specific modification in a second target nucleotide sequence in the plant cell when the Cas endonuclease complexes with the second guide polynucleotide.
A method for generating a plurality of guide RNA molecules for genetic modification in a plant cell, the method comprising providing to the plant cell a polynucleotide expression cassette encoding two or more guide RNA sequences separated by one or more tRNA cleavage sequence, wherein each guide RNA sequence comprises a 3′ spacer sequence that exhibits complementarity to a target sequence in the plant cell, wherein the plant cell's tRNA processing system cleaves a transcript generated from the transcribed polynucleotide expression cassette, thereby generating the plurality of guide RNA sequences.
A nucleic acid expression cassette for generating a plurality of guide RNA molecules for genetic modification in a plant cell comprising a polynucleotide encoding two or more guide RNA sequences separated by one or more tRNA cleavage sequence, wherein each guide RNA sequence comprises a 3′ spacer sequence that exhibits complementarity to a target sequence in the plant cell.
Dependencies are inferred from the claim text.