# US 10,308,947 B2 · Methods and compositions for multiplex RNA guided genome editing and other RNA technologies

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- Number: US 10,308,947 B2
- Document ID: US10308947B2
- Status: Granted
- Kind: B2
- Application number: 14/885,140
- Filed: 2015-10-16
- Published: 2016-09-15
- Granted: 2019-06-04
- Earliest claimed: 2014-10-17
- Assignees: The Penn State Research Foundation
- Applicants: The Penn State Research Foundation
- Inventors: Yinong Yang; Kabin Xie
- Web page: https://patentgrep.com/patent/US10308947B2

## CPC

- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- 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 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/85: for animal cells
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/51: in polymeric form, e.g. multimers, concatemers
- C12N 2330/31: Libraries, arrays
- C12N 2330/51: Specially adapted vectors
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention includes materials and methods to generate numerous small RNAs from one polynucleotide construct (synthetic gene) to facilitate RNA-guided multiplex genome editing, modification, inhibition of expression and other RNA-based technologies. The synthetic gene/polynucleotide construct encodes polycistronic RNA components separated by tRNAs, and preferably also includes regulatory components such as a promoter or terminator to form an expression cassette. Once transcribed in a cell, the transcript is processed by the cell to multiple RNA molecules by the endogenous tRNA processing system. The system can be used for any RNA based gene manipulation method including RNA-mediated genome editing, artificial microRNA mediated gene silencing, small RNA mediated genetic manipulation, double-stranded RNA mediated gene silencing, antisense mechanisms and the like.

## Claims

### Claim 1

A method for producing multiplex RNA mediated genetic manipulation in a recipient cell comprising:
obtaining a polynucleotide construct which encodes two or more guide RNA sequences in tandem array with one or more tRNA cleavage sequence, wherein each guide RNA sequence comprises a 5′ spacer sequence that hybridizes to a target sequence; and introducing said polynucleotide construct to said recipient cell in the presence of a CRISPR-associated nuclease, so that said recipient cell's tRNA processing system cleaves the transcript produced from the transcribed polynucleotide construct at the tRNA sequences to release the guide RNA sequences.

### Claim 2 (depends on 1)

The method of claim 1 wherein said guide RNA sequences target multiple sites within a single gene in said recipient cell.

### Claim 3 (depends on 1)

The method of claim 1 wherein said guide RNA sequences target multiple different genes in said recipient cell.

### Claim 4 (depends on 1)

The method of claim 1 wherein said RNA mediated genetic manipulation is RNA guided genome editing, wherein said RNA guided genome editing includes targeted mutation, homology-dependent repair, transcriptional activation and repression, epigenome editing, and/or genomic labeling.

### Claim 5 (depends on 1)

The method of claim 1 wherein said tRNA cleavage sequence includes a pretRNA acceptor stem, a D-loop arm and a TΨC-loop arm.

### Claim 6 (depends on 1)

The method of claim 1 wherein said tRNA cleavage sequence includes an active site for one or more of RNase P and/or RNase Z and/or RNase E.

### Claim 7 (depends on 1)

The method of claim 1 wherein said tRNA cleavage sequence is SEQ ID NO: 13, 188 or 189.

### Claim 8 (depends on 1)

The method of claim 1 wherein said guide RNA further comprises a scaffold RNA sequence.

### Claim 9 (depends on 1)

The method of claim 1 wherein said polynucleotide construct includes a tRNA-guide RNA-tRNA.

### Claim 10 (depends on 1)

The method of claim 1 wherein said recipient cell is a plant cell.

### Claim 11 (depends on 1)

The method of claim 1 wherein said recipient cell is an animal cell.

### Claim 12 (depends on 1)

The method of claim 1 wherein said cell is a microbial cell.

### Claim 13 (depends on 1)

The method of claim 1 wherein said cell is a human cell.

### Claim 14

A nucleic acid construct for multiplex RNA mediated genome manipulation in a recipient cell comprising: two or more guide RNA sequences in tandem array with one or more tRNA cleavage sequence, wherein each guide RNA sequence comprises a 5′ spacer sequence that hybridizes to a target sequence.

### Claim 15 (depends on 14)

The nucleic acid construct of claim 14 wherein said guide RNA sequences target multiple sites within a single gene in said recipient cell.

### Claim 16 (depends on 14)

The nucleic acid construct of claim 14 wherein said guide RNA sequences target multiple different genes in said recipient cell.

### Claim 17 (depends on 14)

The nucleic acid construct of claim 14 wherein said tRNA cleavage sequence includes a pretRNA acceptor stem, a D-loop arm and a TΨC-loop arm.

### Claim 18 (depends on 14)

The nucleic acid construct of claim 14 wherein said tRNA cleavage sequence includes an active site for one or more of RNase P and/or RNase Z and/or RNase E.

### Claim 19 (depends on 14)

The nucleic acid construct of claim 14 wherein said tRNA cleavage sequence is SEQ ID NO: 13, 188 or 189.

### Claim 20 (depends on 14)

The nucleic acid construct of claim 14 wherein said guide RNA further comprises a scaffold RNA sequence.

### Claim 21 (depends on 14)

The nucleic acid construct of claim 14 wherein said nucleic acid construct includes a tRNA-guide RNA-tRNA.

### Claim 22 (depends on 14)

The nucleic acid construct of claim 14 further comprising regulatory element for expression of said nucleic acid in said recipient cell.

### Claim 23 (depends on 22)

The nucleic acid construct of claim 22 wherein said construct comprises a promoter sequence operably linked to said guide RNA sequences and said tRNA cleavage sequence.

### Claim 24 (depends on 14)

The nucleic acid construct of claim 14 further comprising a terminator sequence.

### Claim 25 (depends on 23)

The nucleic acid construct of claim 23 wherein said promoter sequence is a Pol III promoter.

### Claim 26 (depends on 24)

The nucleic acid construct of claim 24 wherein said terminator sequence is a Pol III terminator.

### Claim 27 (depends on 14)

An expression cassette comprising the nucleic acid construct of claim 14.

### Claim 28 (depends on 27)

A vector comprising the expression cassette of claim 27.

### Claim 29 (depends on 14)

A recipient cell comprising the nucleic acid construct of claim 14.

### Claim 30 (depends on 14)

A genetically modified cell comprising a genomic insertion or deletion said cell having been edited by the introduction of the construct of claim 14.

### Claim 31 (depends on 30)

The cell of claim 30 wherein said cell is a plant cell.

### Claim 32 (depends on 30)

The cell of claim 30 wherein said cell is an animal cell.

### Claim 33 (depends on 30)

The cell of claim 30 wherein said cell is a microbial cell.

### Claim 34 (depends on 30)

The cell of claim 30 wherein said cell is a human cell.