# US 11,898,270 B2 · Pig genome-wide specific sgRNA library, preparation method therefor and application thereof

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- Number: US 11,898,270 B2
- Document ID: US11898270B2
- Status: Granted
- Kind: B2
- Application number: 16/628,481
- Filed: 2017-08-21
- Published: 2020-10-15
- Granted: 2024-02-13
- Earliest claimed: 2017-07-03
- Assignees: Huazhong Agricultural University
- Applicants: Huazhong Agricultural University
- Inventors: Shuhong Zhao; Shengsong Xie; Changzhi Zhao; Xinyun Li; Xiangdong Liu; Xiaosong Han; Gaojuan Yang; Yang Gao; Yilong Chen; Xiaoyong Du; Yiliang Miao; Yunlong Ma; Xiaolei Liu
- Web page: https://patentgrep.com/patent/US11898270B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C40B 40/02: Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- 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 15/85: for animal cells
- C12N 15/907: {in mammalian cells}
- C12Q 1/6869: Methods for sequencing
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/12: in functional genomics, i.e. for the determination of gene function
- C12N 2330/31: Libraries, arrays
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2810/10: Vectors comprising a non-peptidic targeting moiety
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Provided is a pig genome-wide specific sgRNA library, a preparation method therefor, and an application thereof. The sgRNA is targeted at a pig genome-wide protein-coding gene, lincRNA and/or miRNA. Specifically, an sgRNA construct has the following structure: AL-N20-AR, wherein AL is the left homology arm sequence located at the upstream of the coding sequence of a pig specific SgRNA, N20 is the coding sequence of the pig specific SgRNA, and AR is a right homology arm sequence located at the downstream of the coding sequence of the pig specific sgRNA. The sgRNA library can be used for screening functional genes of a pig or for preparing a kit.

## Claims

### Claim 1

A porcine whole genome-specific sgRNA library, wherein the library includes:
(i) N kinds of vectors expressing porcine-specific sgRNA, wherein N is a positive integer ≥20,000, and the porcine-specific sgRNA is a target gene for the porcine whole genome and the target gene is selected from the group consisting of: (a) a protein encoding gene, (b) a lincRNA, (c) a miRNA and (d) a combination of (a), (b) and (c);
in addition, the porcine-specific sgRNA has the following structural characteristics:
(1) the target gene locus targeted by the sgRNA contains PAM as NGG (wherein N is any base of A, T, C, G);
(2) the length of sgRNA is 19 or 20 nt;
(3) the GC content of sgRNA is 40-60%;
(4) a whole-genome off-target evaluation of sgRNA selects off-target sites that do not contain 1 and 2 base mismatches, compared with other sgRNAs, sgRNAs with a smallest number of off-targets are selected;
(5) for a porcine-specific sgRNA that targets a protein-coding gene, the binding position thereof is located in a region 500 bp downstream of the start codon ATG of the coding gene; for a porcine-specific sgRNA that targets a lincRNA gene, the binding position thereof is located in a 500 bp region downstream of the lincRNA transcription start point; for a porcine-specific sgRNA that targets a miRNA gene, the binding position thereof is a miRNA precursor sequence or a mature sequence; and (ii) optionally, M kinds of negative control vectors, which are the vectors expressing unrelated sgRNA, wherein M is a positive integer.

### Claim 2 (depends on 1)

The sgRNA library of claim 1, wherein the expression vector contains an expression cassette expressing porcine sgRNA, having a structure from 5′ to 3′ shown by Formula I:
Z0-Z1-Z2 (I)
wherein Z0 is a first promoter;
Z1 is AL-N20-AR, wherein AL is a left arm sequence upstream of the coding sequence of porcine-specific sgRNA; N20 is a coding sequence of porcine-specific sgRNA; AR is a right arm sequence downstream of the coding sequence of porcine-specific sgRNA;
Z2 is a sgRNA scaffold sequence.

### Claim 3 (depends on 2)

The sgRNA library of claim 2, wherein the expression cassette has a structure from 5′ to 3′ shown in Formula II:
Z0-Z1-Z2-L-Z3-Z4 (II)
wherein Z0, Z1, and Z2 are as described above,
L is an optional interval sequence;
Z3 is a second promoter;
Z4 is a reporter gene.

### Claim 4 (depends on 2)

The sgRNA library of claim 2, wherein the first promoter is a promoter that starts sgRNA expression.

### Claim 5 (depends on 3)

The sgRNA library of claim 3, wherein the second promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, a CBh promoter, a tissue-specific promoter, and a combination thereof.

### Claim 6 (depends on 2)

The sgRNA library of claim 2, wherein Z1 has a structure of formula (IV):
5′-TATCTTGTGGAAAGGACGAAACACCG (SEQ ID NO: 163)
N20GTTTTAGAGCTAGAAATAGCAAGTTAAAAT (SEQ ID NO: 164)-3′, wherein N20 is a coding sequence of a porcine-specific sgRNA.

### Claim 7 (depends on 1)

A kit containing:
(a) a first container and a porcine whole genome-specific sgRNA library of claim 1 located in the container; and
(b) the instructions describing the instructions for using the porcine whole genome-specific sgRNA library.

### Claim 8 (depends on 7)

The kit of claim 7, wherein the kit further includes:
(c) a second container and a porcine cell located in the second container, the porcine cell contains a constitutive or inducible protein expressing Cas9.