# US 11,306,308 B2 · High-throughput CRISPR-based library screening

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- Number: US 11,306,308 B2
- Document ID: US11306308B2
- Status: Granted
- Kind: B2
- Application number: 15/775,621
- Filed: 2016-11-11
- Published: 2018-11-15
- Granted: 2022-04-19
- Earliest claimed: 2015-11-13
- Assignees: Massachusetts Institute of Technology; The Brigham and Women's Hospital, Inc.
- Applicants: Massachusetts Institute of Technology; The Brigham and Women's Hospital, Inc.
- Inventors: David K. Gifford; Nisha Rajagopal; Richard Irving Sherwood
- Web page: https://patentgrep.com/patent/US11306308B2

## CPC

- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/102: {Mutagenizing nucleic acids}
- 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 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/531: Stem-loop; Hairpin
- 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

Provided herein is an improved method for performing CRISPR/Cas based screening that is not dependent on viral cloning methods.

## Claims

### Claim 1

A multiplexed editing regulatory assay (MERA) construct comprising: one or more copies of a defective gRNA expression construct wherein each defective gRNA expression construct comprises:
i. a dummy gRNA that is not homologous to a eukaryotic genome, and is unable to target a Cas nuclease to the genome for a double strand break,
ii. a guide hairpin sequence,
iii. a native regulatory region which will be targeted using CRISPR/Cas endonuclease; and
iv. an operably linked promoter.

### Claim 2 (depends on 1)

The MERA construct of claim 1, wherein the eukaryotic genome is a human genome.

### Claim 3 (depends on 1)

The MERA construct of claim 1, wherein the gRNA is 19-21 nucleotides in length.

### Claim 4 (depends on 1)

The MERA construct of claim 1, wherein the guide hairpin sequence is about 40 nucleotides in length and once transcribed can be bound to a CRISPR/Cas nuclease.

### Claim 5 (depends on 1)

A host cell comprising the MERA construct of claim 1.

### Claim 6 (depends on 5)

The host cell of claim 5, wherein a single defective gRNA expression construct is integrated into the host cell genome.

### Claim 7 (depends on 5)

The host cell of claim 5, wherein 2-10 defective gRNA expression constructs are integrated into the host cell genome.

### Claim 8 (depends on 5)

A population of host cells of claim 5.

### Claim 9 (depends on 5)

The host cell of claim 5, further comprising a CRISPR/Cas nuclease and/or a coding sequence for the CRISPR/Cas nuclease.

### Claim 10 (depends on 5)

The host cell of claim 5, further comprising Cas9 nuclease.

### Claim 11 (depends on 9, 10)

The host cell of claim 9 or 10, further comprising a library of exogenous gRNA homology fragments spanning across the targeted native regulatory region, optionally wherein the gRNA homology fragment further comprises a coding sequence for a reporter protein or a reporter fusion protein.

### Claim 12 (depends on 11)

A population of cells of claim 11, wherein each cell has integrated into its genome one or more functional gRNA expression construct at the dummy guide locus.

### Claim 13 (depends on 12)

The population of cells of claim 12, wherein the genomic integration is in a virus-independent manner.

### Claim 14 (depends on 1)

A method comprising:
(1) introducing into a host cell comprising the MERA construct of claim 1: a library of exogenous gRNA homology fragments spanning across the targeted native regulatory region, wherein the gRNA homology fragment further comprises a coding sequence for a reporter protein or a reporter fusion protein, and
(2) allowing a single exogenous gRNA homology fragment to homologously recombine with the dummy gRNA construct in the presence of a CRISPR/Cas nuclease to form a functional guide RNA construct, wherein the host cell comprises a reporter gene coding sequence integrated into its genome.