# US 11,078,483 B1 · Methods for measuring and improving CRISPR reagent function

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- Number: US 11,078,483 B1
- Document ID: US11078483B1
- Status: Granted
- Kind: B1
- Application number: 15/694,452
- Filed: 2017-09-01
- Granted: 2021-08-03
- Earliest claimed: 2016-09-02
- Assignees: KSQ Therapeutics, Inc.
- Applicants: KSQ Therapeutics, Inc.
- Inventors: Gregory V. Kryukov; Michael R. Schlabach; Jason J. Merkin
- Web page: https://patentgrep.com/patent/US11078483B1

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- 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}
- 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
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids
- C12N 2330/31: Libraries, arrays
- C12N 2330/50: Biochemical production, i.e. in a transformed host cell
- G16B 35/10: Design of libraries
- 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)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The invention describes a novel system for identifying optimized gRNAs for use in CRISPR/Cas9 genome editing platforms. The invention allows for the determination of specific gene alterations rendered by a particular gRNA, thereby permitting the generation of optimized gRNA libraries.

## Claims

### Claim 1

A method of preparing an optimized guide RNA (gRNA) library comprising;
(a) introducing a library of nucleic acid constructs into a population of cells modified to express a Cas9 protein, wherein each construct comprises a gRNA sequence and a sensor sequence, wherein the sensor sequence comprises the corresponding gRNA target sequence;
(b) culturing said cells from step (a) under conditions permitting the expression of said gRNA and said Cas9 protein;
(c) amplifying the nucleic acid constructs sequences by polymerase chain reaction (PCR) from the cells of step (b) to obtain a plurality of amplicons, wherein each amplicon comprises the gRNA sequence and the sensor sequence;
(d) sequencing said plurality of amplicons;
(e) determining a pattern of alterations in the sensor sequence in each of the amplicons; and
(f) identifying a population of gRNAs that produce a desired pattern of alterations in the sensor sequence,
wherein the population of cells is modified with a nucleic acid encoding the Cas9 protein, and
wherein the nucleic acid encoding the Cas9 protein mRNA is introduced into the population of cells by electroporation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the library of nucleic acid constructs encode a genome wide or a sub-genome wide library of gRNAs.

### Claim 3 (depends on 1)

The method of claim 1, wherein the nucleic acid encoding a Cas9 protein is encoded by a viral vector.

### Claim 4 (depends on 3)

The method of claim 3, wherein the viral vector is a lentiviral vector and is introduced at a titer of at least about 1×106 infectious particles/mL.

### Claim 5 (depends on 1)

The method of claim 1, wherein the Cas9 protein is a dCas9 protein.

### Claim 6 (depends on 5)

The method of claim 5, wherein the dCas9 protein is fused with a transcriptional repressor domain.

### Claim 7 (depends on 5)

The method of claim 5, wherein the dCas9 protein is fused with a transcriptional activator.

### Claim 8 (depends on 5)

The method of claim 5, wherein the dCas9 protein is fused with a heterologous protein domain.

### Claim 9 (depends on 1)

The method of claim 1, wherein the Cas9 protein is a nickase mutant of Cas9.

### Claim 10 (depends on 1)

The method of claim 1, wherein the expression of the nucleic acid encoding a Cas9 protein is under the control of an inducible gene element.

### Claim 11 (depends on 1)

The method of claim 1, wherein the population of cells are mammalian cells.

### Claim 12 (depends on 1)

The method of claim 1, wherein the sensor sequence is a nucleic acid sequence from a mammalian genome.

### Claim 13 (depends on 12)

The method of claim 12, wherein the sensor sequence is from a human genome and the gRNAs identified in step (e) produce the desired pattern of alterations in the human sensor sequence.

### Claim 14 (depends on 1)

The method of claim 1, wherein the nucleic acid construct is encoded by a lentiviral vector, and wherein the lentiviral vector is introduced at a titer of at least about 1×106 infectious particles/mL.

### Claim 15 (depends on 1)

The method of claim 1, wherein the nucleic acid construct is under the control of an inducible gene element.

### Claim 16 (depends on 1)

The method of claim 1, wherein the sequencing comprises high throughput sequencing.

### Claim 17 (depends on 1)

The method of claim 1, wherein the desired pattern of alterations is selected from a group consisting of insertions in the sensor sequence, deletions in the sensor sequence, and mutations in the sensor sequence.

### Claim 18 (depends on 1)

The method of claim 1, further comprising introducing a nucleic acid sequence encoding a repair template into the transduced population of cells.

### Claim 19 (depends on 1)

The method of claim 1, wherein the desired pattern of alterations are a result of a mechanism selected from the group consisting of non-homologous end-joining (NHEJ) and homology-directed repair (HDR).

### Claim 20 (depends on 1)

The method of claim 1, further comprising;
(g) amplifying an endogenous target nucleic acid sequence by PCR from the cells of step (c) to obtain a plurality of endogenous target amplicons;
(h) sequencing said endogenous target amplicons;
(i) determining a pattern of alterations in said endogenous target sequence;
(j) comparing the pattern of alterations in the endogenous target sequence with the pattern of alterations in the sensor sequence; and
(k) determining a population of gRNAs that produce a desired pattern of alterations in both the endogenous target sequence and the sensor sequence.

### Claim 21 (depends on 20)

The method of claim 20, wherein the desired pattern of alterations in the endogenous target sequence and the sensor sequence is selected from the group consisting of insertions, deletions, and mutations.

### Claim 22 (depends on 1)

The method of claim 1, further comprising:
(g) quantifying a transcription level of an endogenous target sequence and/or sensor sequence from the population of cells of step (c) by quantitative PCR (qPCR);
(h) comparing the transcription level of step (g) to a transcription level of the endogenous target sequence and/or sensor sequence obtained from a control cell population by qPCR;
(i) determining a pattern of transcriptional changes in the endogenous target sequence and/or sensor sequence; and
(j) determining a population of gRNAs that produce a desired pattern of transcriptional changes in the endogenous target sequence and/or sensor sequence.