# US 12,215,319 B2 · Methods and compositions for targeted genomic insertion

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- Number: US 12,215,319 B2
- Document ID: US12215319B2
- Status: Granted
- Kind: B2
- Application number: 16/634,215
- Filed: 2018-07-26
- Published: 2021-03-25
- Granted: 2025-02-04
- Earliest claimed: 2017-08-04
- Assignees: Syngenta Crop Protection AG
- Applicants: Syngenta Participations AG
- Inventors: Zhongying Chen; Mary-Dell Chilton; Shujie Dong; Qiudeng Que; Heng Zhong
- Web page: https://patentgrep.com/patent/US12215319B2

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to methods and compositions for chimeric RNA comprising a guide RNA and bait RNA for modifying a target site in the genome of a cell. Such modifications include integration of a transgene and allelic mutations and modifications of native genes. Also provided are plants comprising a modified nucleic acid sequence compared to the native gene integrated into a targeted genomic site in the plant genome.

## Claims

### Claim 1

A chimeric RNA for targeted genomic modification of a cell, wherein the chimeric RNA comprises:
a) a guide RNA comprising a polynucleotide targeting guide sequence of at least 8 contiguous nucleotides and at least 80% identical to a target nucleic acid sequence of at least 8 contiguous nucleotides within a target nucleic acid molecule; and
b) a bait RNA comprising at least 8 contiguous nucleic acids, wherein the nucleic acid sequence of the bait RNA is at least 70% complementary to at least 8 contiguous nucleic acids of a donor DNA molecule, wherein the donor DNA molecule is intended for integration into the genome of a cell, wherein a tracrRNA is operably linked to said bait RNA sequence.

### Claim 2 (depends on 1)

The chimeric RNA of claim 1, wherein said bait RNA is operably linked to said guide RNA.

### Claim 3 (depends on 2)

The chimeric RNA of claim 2, wherein the guide RNA further comprises a nucleic acid sequence which is a binding site for a site-directed modifying polypeptide.

### Claim 4 (depends on 1)

The chimeric RNA of claim 1, wherein the guide RNA comprises a crRNA.

### Claim 5 (depends on 4)

The chimeric RNA of claim 4, wherein the crRNA is operably linked to said bait RNA.

### Claim 6 (depends on 1)

The chimeric RNA of claim 1, wherein the guide RNA comprises a crRNA operably linked to said tracrRNA.

### Claim 7 (depends on 1)

The chimeric RNA of claim 1, wherein the guide RNA further comprises a tracrRNA, wherein the tracrRNA interacts with the guide RNA to create an RNA duplex that is a binding site for a site-directed modifying polypeptide.

### Claim 8 (depends on 7)

The chimeric RNA of claim 7, wherein the guide RNA comprises a tracrRNA operably linked to a bait RNA.

### Claim 9 (depends on 1)

An expression cassette comprising a nucleic acid sequence encoding the chimeric RNA of claim 1.