Novel cas9 systems and methods of use
AssigneePioneer Hi-Bred International, Inc.
InventorsANDREW MARK CIGAN, MATTHEW G. KING, HAINING LIN, JOSHUA K. YOUNG
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Abstract
Compositions and methods are provided for novel Cas9 systems, including, but not limiting to, novel guide polynucleotide/Cas9 endonucleases complexes, single or dual guide RNAs, guide RNA elements, and Cas9 endonucleases. The present disclosure also describes methods for genome modification of a target sequence in the genome of a cell, for gene editing, and for inserting a polynucleotide of interest into the genome of a cell. Also provided are nucleic acid constructs and cells having an altered target site or altered polynucleotide of interest produced by the methods described herein.
Claims19
A synthetic composition comprising a target sequence and a single guide RNA capable of forming a guide RNA/Cas9 endonuclease complex, wherein said guide RNA/Cas9 endonuclease complex can recognize, bind to, and optionally nick or cleave the target sequence, wherein said single guide RNA comprises SEQ ID NO:
- 188, a functional fragment of SEQ ID NO:
- 188, or a functional variant of SEQ ID NO:
- 188.
A synthetic composition comprising a target sequence and a single guide RNA capable of forming a guide RNA/Cas9 endonuclease complex, wherein said guide RNA/Cas9 endonuclease complex can recognize, bind to, and optionally nick or cleave the target sequence, wherein said single guide RNA comprises a chimeric non-naturally occurring crRNA linked to a tracrRNA, wherein said tracrRNA comprises SEQ ID NO:
- 142 or 165, a functional fragment of SEQ ID NO:
- 142 or165, or a functional variant of SEQ ID NO:
- 142 or 165.
A synthetic composition comprising a target sequence and a single guide RNA capable of forming a guide RNA/Cas9 endonuclease complex, wherein said guide RNA/Cas9 endonuclease complex can recognize, bind to, and optionally nick or cleave the target sequence, wherein said single guide RNA comprises a chimeric non-naturally occurring crRNA linked to a tracrRNA, wherein said chimeric non-naturally occurring crRNA comprises SEQ ID NO:
- 119, a functional fragment of SEQ ID NO:
- 119, or a functional variant of SEQ ID NO:
- 119.
A synthetic composition comprising a target sequence and a guide RNA capable of forming a guide RNA/Cas9 endonuclease complex, wherein said guide RNA/Cas9 endonuclease complex can recognize, bind to, and optionally nick or cleave the target sequence, wherein said guide RNA is a duplex molecule comprising a chimeric non-naturally occurring crRNA and a tracrRNA, wherein said chimeric non-naturally occurring crRNA comprises a variable targeting domain capable of hybridizing to said target sequence, wherein said tracrRNA comprises SEQ ID NO:
- 142 or 165, a functional fragment of SEQ ID NO:
- 142 or165, or a functional variant of SEQ ID NO:
- 142 or 165;
- wherein said chimeric non-naturally occurring crRNA comprises a variable targeting domain capable of hybridizing to said target sequence.
A synthetic composition comprising a target sequence and a guide RNA capable of forming a guide RNA/Cas9 endonuclease complex, wherein said guide RNA/Cas9 endonuclease complex can recognize, bind to, and optionally nick or cleave the target sequence, wherein said guide RNA is a duplex molecule comprising a chimeric non-naturally occurring crRNA and a tracrRNA, wherein said chimeric non-naturally occurring crRNA comprises SEQ ID NO:
- 119, a functional fragment of SEQ ID NO:
- 119, or a functional variant of SEQ ID NO:
- 119;
- wherein said chimeric non-naturally occurring crRNA comprises a variable targeting domain capable of hybridizing to said target sequence.
A synthetic composition comprising a target sequence and a guide RNA/Cas9 endonuclease complex comprising a Cas9 endonuclease comprising SEQ ID NO:
- 50, a functional fragment of SEQ ID NO:
- 50, or a functional variant of SEQ ID NO:
- 50;
- and at least one guide RNA, wherein said guide RNA/Cas9 endonuclease complex is capable of recognizing, binding to, and optionally nicking or cleaving all or part of the target sequence.
A synthetic composition comprising a target sequence and a guide RNA/Cas9 endonuclease complex comprising at least one guide RNA and a Cas9 endonuclease, wherein said Cas9 endonuclease is encoded by a DNA sequence comprising SEQ ID NO:
- 27, a functional fragment of SEQ ID NO:
- 27, or a functional variant SEQ ID NO:
- 27, wherein said guide RNA/Cas9 endonuclease complex is capable of recognizing, binding to, and optionally nicking or cleaving all or part of the target sequence.
A method for modifying a target site in the genome of a cell, the method comprising introducing into said cell at least one guide RNA and at least one Cas9 endonuclease comprising SEQ ID NO:
- 50, a functional fragment of SEQ ID NO:
- 50, or a functional variant of SEQ ID NO:
- 50;
- wherein said guide RNA and Cas9 endonuclease can form a complex that is capable of recognizing, binding to, and optionally nicking or cleaving all or part of said target site;
- further comprising identifying at least one cell that has a modification at said target, wherein the modification at said target site is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).
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Dependencies are inferred from the claim text.