# US 11,299,731 B1 · CRISPR editing to embed nucleic acid landing pads into genomes of live cells

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- Number: US 11,299,731 B1
- Document ID: US11299731B1
- Status: Granted
- Kind: B1
- Application number: 17/475,267
- Filed: 2021-09-14
- Published: 2022-03-24
- Granted: 2022-04-12
- Earliest claimed: 2020-09-15
- Assignees: Inscripta, Inc.
- Applicants: Inscripta, Inc.
- Inventors: Daniel Held
- Web page: https://patentgrep.com/patent/US11299731B1

## CPC

- 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 15/102: {Mutagenizing nucleic acids}
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 15/907: {in mammalian cells}
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C12N 15/85: for animal cells
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- 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)
- 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)

## Abstract

The present disclosure relates to compositions, methods, modules and automated integrated instrumentation for multiplex delivery of “landing pad” edits into the genomes of a population of live cells. The landing pads then may be leveraged to insert very large DNA sequences into the genomes of the population of live cells.

## Claims

### Claim 1

A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of:
designing and synthesizing a library of editing cassettes comprising landing pads, wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad;
inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors;
transforming the population of cells with the library of editing vectors;
allowing editing to take place in the population of cells to produce edited cells;
transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for a recombinase or a meganuclease under control of an inducible promoter;
inducing expression of the recombinase or meganuclease to insert the large DNA payloads into the landing pads; and
screening for cells comprising the desired phenotype or genotype.

### Claim 2 (depends on 1)

The method of claim 1, wherein the vectors carrying large DNA payloads comprise a coding sequence for a recombinase, the landing pads comprise a recognition sequence for the recombinase and the large DNA payloads comprise recognition sequences for the recombinase flanking the large DNA payload.

### Claim 3 (depends on 2)

The method of claim 2, wherein the recombinase is a cyclization recombination enzyme (Cre) and the landing pad and large DNA payload comprise lox recombination sites.

### Claim 4 (depends on 2)

The method of claim 2, wherein the recombinase is flippase and the landing pad and large DNA payload comprise flippase recognition targets (FRTs).

### Claim 5 (depends on 1)

The method of claim 1, wherein the vectors carrying large DNA payloads comprise a coding sequence for a meganuclease, the landing pads comprise a recognition sequence for the meganuclease, and the large DNA payloads further comprise homologous recombination sequences flanking the DNA playloads.

### Claim 6 (depends on 5)

The method of claim 5, wherein the meganuclease belongs to the LAGLIDADG family of nucleases.

### Claim 7 (depends on 6)

The method of claim 6, wherein the meganuclease is I-SceI.

### Claim 8 (depends on 6)

The method of claim 6, wherein the meganuclease is I-CreI.

### Claim 9 (depends on 6)

The method of claim 6, wherein the meganuclease is I-DmoI.

### Claim 10 (depends on 1)

The method of claim 1, wherein the editing cassettes further comprise a barcode.

### Claim 11 (depends on 1)

The method of claim 1, wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette.

### Claim 12 (depends on 1)

The method of claim 1, wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the allowing step and the transforming the editing cells step.

### Claim 13 (depends on 12)

The method of claim 12, wherein the selectable marker in the vectors carrying the large DNA payloads is different from a selectable marker in the editing cassettes.

### Claim 14 (depends on 1)

The method of claim 1, wherein the vectors carrying the large DNA payloads comprise the coding sequence of the recombinase or meganuclease under the control of an inducible promoter.

### Claim 15 (depends on 14)

The method of claim 14, wherein the inducible promoter is a pL promoter.

### Claim 16 (depends on 14)

The method of claim 14, wherein the inducible promoter is a pBAD promoter.

### Claim 17 (depends on 1)

The method of claim 1, wherein the vectors carrying large DNA payloads further comprise an origin of replication and a selectable marker.

### Claim 18 (depends on 1)

The method of claim 1, wherein the large DNA payloads are from 100 bp to 100 Kb in length.

### Claim 19 (depends on 18)

The method of claim 18, wherein the large DNA payloads are from 500 bp to 50 Kb in length.

### Claim 20 (depends on 1)

The method of claim 1, wherein the screening step comprises polymerase chain reaction (PCR) analysis with appropriate primer sets; a metabolic test; measurement of transcript level; a phenotypic assay; detection of a protein product using an antibody specific to the protein product;
or DNA sequencing of the integrated large DNA payload.

### Claim 21

A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of:
designing and synthesizing a library of editing cassettes comprising landing pads wherein the landing pads comprise a recognition sequence for a recombinase and wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad;
inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors;
transforming the population of cells with the library of editing vectors;
allowing editing to take place in the population of cells to produce edited cells;
transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for the recombinase under control of an inducible promoter;
inducing expression of the recombinase to insert the large DNA payloads into the landing pads; and
screening for cells comprising the desired phenotype or genotype.

### Claim 22 (depends on 21)

The method of claim 21, wherein the recombinase is a cyclization recombination enzyme (Cre) and the landing pad and large DNA payload comprise lox recombination sites.

### Claim 23 (depends on 21)

The method of claim 21, wherein the recombinase is flippase and the landing pad and large DNA payload comprise flippase recognition targets (FRTs).

### Claim 24 (depends on 21)

The method of claim 21, wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette and wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the allowing step and the transforming the editing cells step.

### Claim 25

A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of:
designing and synthesizing a library of editing cassettes comprising landing pads wherein the landing pads comprise a recognition sequence for a meganuclease and wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad;
inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors;
transforming the population of cells with the library of editing vectors;
allowing editing to take place in the population of cells to produce edited cells;
transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for the meganuclease under control of an inducible promoter;
inducing expression of the meganuclease to insert the large DNA payloads into the landing pads; and
screening for cells comprising the desired phenotype or genotype.

### Claim 26 (depends on 25)

The method of claim 25, wherein the meganuclease belongs to the LAGLIDADG family of nucleases.

### Claim 27 (depends on 26)

The method of claim 26, wherein the meganuclease is I-SceI.

### Claim 28 (depends on 26)

The method of claim 26, wherein the meganuclease is I-CreI.

### Claim 29 (depends on 26)

The method of claim 26, wherein the meganuclease is I-DmoI.

### Claim 30 (depends on 25)

The method of claim 25, wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette and wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the allowing step and the transforming the editing cells step.