# US 12,215,364 B2 · CRISPR-cas systems for genome editing

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- Number: US 12,215,364 B2
- Document ID: US12215364B2
- Status: Granted
- Kind: B2
- Application number: 17/154,445
- Filed: 2021-01-21
- Published: 2021-06-03
- Granted: 2025-02-04
- Earliest claimed: 2018-12-14
- Assignees: Pioneer Hi-Bred International, Inc.
- Applicants: Pioneer Hi-Bred International, Inc.
- Inventors: Zhenglin Hou; Tautvydas Karvelis; Virginijus Siksnys; Joshua K Young
- Web page: https://patentgrep.com/patent/US12215364B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/82: for plant cells {, e.g. plant artificial chromosomes (PACs)}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/902: {using homologous recombination}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Compositions and methods are provided for genome modification of a target sequence in the genome of a cell, using a novel Cas endonuclease. The methods and compositions employ a guide polynucleotide/endonuclease system to provide an effective system for modifying or altering target sequences within the genome of a cell or organism. Also provided are novel effectors and endonuclease systems and elements comprising such systems, such as guide polynucleotide/endonuclease systems comprising an endonuclease. Compositions and methods are also provided for guide polynucleotide/endonuclease systems comprising at least one endonuclease, optionally covalently or non-covalently linked to, or assembled with, at least one additional protein subunit, and for compositions and methods for direct delivery of endonucleases as ribonucleotide proteins.

## Claims

### Claim 1

A synthetic composition comprising:
(a) a Cas endonuclease or a functional fragment thereof, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 38 or SEQ ID NO:368, wherein the Cas endonuclease or the functional fragment comprises a tri-split RuvC domain, at least one zinc finger motif, and the following amino acid motifs: GxxxG, ExL, Cx1C, and Cxn(C or H); wherein G=Glycine, E=Glutamate, C=Cysteine, H=Histidine, L=Leucine, x=any amino acid and wherein n=an integer between 0 and 11, and wherein the Cas endonuclease or the functional fragment thereof recognizes a PAM sequence comprising a NNNNCCD nucleotide sequence, wherein N=cytosine, thymine, adenine, or guanine; C=cytosine; and D=thymine, adenine, or guanine;
(b) a target double-stranded DNA polynucleotide, wherein the target double-stranded DNA polynucleotide is heterologous to the source of the Cas endonuclease,
(c) a guide polynucleotide comprising a variable targeting domain that comprises a region of complementarity to the target double-stranded DNA polynucleotide; wherein the Cas endonuclease or the functional fragment thereof recognizes the PAM sequence on the target double-stranded DNA polynucleotide, wherein the guide polynucleotide and the Cas endonuclease or the functional fragment thereof, form a guide polynucleotide-Cas endonuclease complex that binds to the target double-stranded DNA polynucleotide, wherein the Cas endonuclease or the functional fragment thereof comprises DNA binding activity,
wherein the guide polynucleotide-Cas endonuclease complex is heterologous to Clostridium novyi.

### Claim 2 (depends on 1)

The synthetic composition of claim 1, wherein the Cas endonuclease is provided as a polynucleotide encoding the Cas endonuclease.

### Claim 3 (depends on 1)

The synthetic composition of claim 1, wherein the guide polynucleotide-Cas endonuclease complex enables the Cas endonuclease to induce double-strand cleavage of the target double-stranded DNA polynucleotide.

### Claim 4 (depends on 1)

The synthetic composition of claim 1, further comprising an expression element.

### Claim 5 (depends on 1)

The synthetic composition of claim 1, further comprising a transgene.

### Claim 6 (depends on 1)

The synthetic composition of claim 1, further comprising a donor DNA molecule.

### Claim 7 (depends on 1)

The synthetic composition of claim 1, further comprising a polynucleotide modification template.

### Claim 8

A synthetic composition comprising:
(a) a Cas protein or functional fragment thereof, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 38 or SEQ ID NO:368, wherein the Cas protein or the functional fragment thereof comprises a tri-split RuvC domain and at least one zinc finger motif, and wherein the Cas protein or the functional fragment thereof recognizes a PAM sequence comprising a NNNNCCD nucleotide sequence, wherein N=cytosine, thymine, adenine, or guanine; C=cytosine; and D=thymine, adenine, or guanine;
(b) a target double-stranded DNA polynucleotide that comprises the PAM sequence, wherein the target double-stranded DNA polynucleotide is heterologous to the source of the Cas protein; and
(c) a guide polynucleotide comprising a variable targeting domain that comprises a region of complementarity to the target double-stranded DNA polynucleotide;
wherein the Cas protein recognizes the PAM sequence on the target double-stranded DNA polynucleotide, wherein the guide polynucleotide and the Cas protein form a complex that binds to the target double-stranded DNA polynucleotide; wherein the Cas protein comprises DNA binding activity and lacks double-strand DNA cleavage activity.

### Claim 9 (depends on 1)

The synthetic composition of claim 1, further comprising a deaminase.

### Claim 10 (depends on 1)

The synthetic composition of claim 1, wherein the Cas endonuclease is part of a fusion protein.

### Claim 11 (depends on 10)

The synthetic composition of claim 10, wherein the fusion protein further comprises a nuclease domain.

### Claim 12 (depends on 1)

The synthetic composition of claim 1, further comprising a eukaryotic cell.

### Claim 13 (depends on 12)

The synthetic composition of claim 12, wherein the eukaryotic cell is a plant cell, an animal cell, or a fungal cell.

### Claim 14 (depends on 13)

The synthetic composition of claim 13, wherein the plant cell is a monocot cell or a dicot cell.

### Claim 15 (depends on 13)

The synthetic composition of claim 13, wherein the plant cell is from an organism selected from the group consisting of: maize, soybean, cotton, wheat, canola, oilseed rape, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.

### Claim 16 (depends on 1)

The synthetic composition of claim 1, wherein at least one component is attached to a solid matrix.

### Claim 17

A method of introducing a targeted edit in a target polynucleotide, the method comprising:
providing a composition comprising:
(a) a Cas endonuclease or a functional fragment thereof, comprising a polypeptide at least 90% identical to SEQID NO: 38 or SEQ ID NO:368, wherein the Cas endonuclease or functional fragment comprises at least one zinc finger domain, a tri-split RuvC domain, and the following amino acid motifs: GxxxG, ExL, CxnC, and Cxn(C or H);
wherein G=Glycine, E=Glutamate, C=Cysteine, H=Histidine, L=Leucine, x=any amino acid and wherein n=an integer between 0 and 11, and wherein the Cas endonuclease or the functional fragment thereof recognizes a PAM sequence comprising a NNNNCCD nucleotide sequence, wherein N=cytosine, thymine, adenine, or guanine; C=cytosine; and D=thymine, adenine, or guanine, wherein the Cas endonuclease recognizes the PAM sequence on the target polynucleotide, and wherein the Cas endonuclease comprises DNA binding activity; and
(b) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a portion of the target polynucleotide, wherein the guide polynucleotide and Cas endonuclease form a guide polynucleotide-Cas endonuclease complex that can recognize and bind to the target polynucleotide,
wherein the guide polynucleotide-Cas endonuclease complex is heterologous to Clostridium novyi.

### Claim 18 (depends on 17)

The method of claim 17, further comprising a cell, the method further comprising introducing at least one nucleotide modification in the genome of at least one cell of the organism as compared to the target sequence of the genome of the cell prior to the introduction of the composition, further comprising incubating the cell and generating a whole organism from the cell, and ascertaining the presence of the at least one nucleotide modification in the genome of at least one cell of the organism as compared to the target sequence of the genome of the cell prior to the introduction of the composition.

### Claim 19 (depends on 18)

A progeny of the organism obtained by the method of claim 18, wherein the progeny comprises the Cas endonuclease or functional fragment thereof.

### Claim 20 (depends on 18)

The method of claim 18, wherein the cell is a eukaryotic cell.

### Claim 21 (depends on 20)

The method of claim 20, wherein the eukaryotic cell is derived or obtained from an animal, a fungus, or a plant.

### Claim 22 (depends on 21)

The method of claim 21, wherein the plant is a monocot or a dicot.

### Claim 23 (depends on 21)

The method of claim 21, wherein the plant is selected from the group consisting of: maize, soybean, cotton, wheat, canola, oilseed rape, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.

### Claim 24 (depends on 17)

The method of claim 17, further comprising introducing either a donor DNA molecule, or a polynucleotide modification template.