# US 11,898,148 B2 · Sequential loadings of multiple delivery vectors using a single selectable marker > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,898,148 B2 - Document ID: US11898148B2 - Status: Granted - Kind: B2 - Application number: 16/092,828 - Filed: 2017-04-12 - Published: 2020-05-21 - Granted: 2024-02-13 - Earliest claimed: 2016-04-12 - Assignees: CarryGenes Bioengineering, LLC - Applicants: SynPloid Biotek, LLC - Inventors: Edward Perkins; Amy Greene - Web page: https://patentgrep.com/patent/US11898148B2 ## CPC - C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host - C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease - C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) - C12N 15/65: using markers (enzymes used as markers C12N15/52) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention provides a novel method for targeted integration of nucleic acids onto an autonomously replicating nucleic acid using site-directed recombination that allows for sequential loading of multiple delivery vectors using a single selectable marker. ## Claims ### Claim 1 A method for sequential loading of multiple nucleic acid sequences, each nucleic acid sequence containing at least one gene of interest onto an autonomously replicating nucleic acid sequence having multiple site-specific recombination target sites by removal and reuse of a single recyclable selectable marker gene cassette having an unlimited number of recyclings, the method comprising: in a population of animal or plant cells carrying the autonomously replicating nucleic acid sequence, using a first recombination system, to integrate a first delivery vector comprising a recyclable marker gene cassette and a first gene of interest into a first of the multiple site-specific recombination target sites on, the autonomously replicating nucleic acid sequence; with a second recombination system and using signal sites alpha, excising the recyclable marker gene cassette from the autonomously replicating nucleic acid sequence, wherein the first gene of interest remains on the autonomously replicating nucleic acid sequence; identifying a first group of cells from the population, wherein the first gene of interest has been loaded onto the autonomously replicating nucleic acid sequence selectable and the recyclable marker gene cassette is no longer present; in the identified first group of cells, using the first recombination system, integrating to integrate a second delivery vector comprising the recyclable marker gene cassette and a second gene of interest into a second of the multiple site-specific recombination target sites on the autonomously replicating nucleic acid sequence; and with the second recombination system using signal sites beta, excising the recyclable marker gene cassette from the autonomously replicating nucleic acid sequence, wherein the second gene of interest remains on the autonomously replicating nucleic acid sequence. ### Claim 2 (depends on 1) The method of claim 1, further comprising the steps of: identifying a second group of cells in which the first and the second genes of interest have been loaded onto the autonomously replicating nucleic acid sequence and the recyclable marker gene cassette is no longer present; in the identified second group of cells, using the first recombination system, integrating a third delivery vector comprising the recyclable marker gene cassette and a third gene of interest into a third of the multiple recombination target sites on the autonomously replicating nucleic acid sequence; and with the second recombination system using signal sites gamma, excising the recyclable marker gene cassette from the autonomously replicating nucleic acid sequence wherein the third gene of interest remains on the autonomously replicating nucleic acid sequence. ### Claim 3 (depends on 1) The method of claim 1, wherein the autonomously replicating nucleic acid sequence is an endogenous chromosome. ### Claim 4 (depends on 1) The method of claim 1, wherein the autonomously replicating nucleic acid sequence is a synthetic chromosome. ### Claim 5 (depends on 4) The method of claim 4, wherein the synthetic chromosome is a mammalian synthetic chromosome. ### Claim 6 (depends on 5) The method of claim 5, wherein the synthetic chromosome is a human synthetic chromosome. ### Claim 7 (depends on 1) The method of claim 1, wherein the first recombination system is a Lambda integrase system. ### Claim 8 (depends on 1) The method of claim 1, wherein the second recombination system is a Cre/lox recombination system. ### Claim 9 (depends on 1) The method of claim 1, wherein the signal sites alpha and beta are selected from Lox2272, Lox5171, LoxM2 and LoxP, and the signal sites alpha and beta are not the same. ### Claim 10 (depends on 1) The method of claim 1, wherein the marker gene is a gene encoding for a fluorescent protein. ### Claim 11 (depends on 1) The method of claim 1, wherein each gene of interest is tagged with a different fluorescent marker. ### Claim 12 A method for sequential loading of multiple nucleic acid sequences, each nucleic acid sequence containing at least one gene of interest, onto an autonomously replicating synthetic chromosome having multiple site-specific recombination target sites by removal and reuse of a single recyclable selectable marker gene cassette having an unlimited number of recyclings, the method comprising: in a population of animal or plant cells carrying the autonomously replicating synthetic chromosome, using a first recombination system to integrate a first delivery vector comprising a recyclable marker gene cassette and a first gene of interest into a first of the multiple recombination target sites on the autonomously replicating synthetic chromosome; with a second recombination system using signal sites alpha, excising the recyclable marker gene cassette from the autonomously replicating synthetic chromosome, wherein the first gene of interest remains on the autonomously replicating synthetic chromosome; identifying a first group of cells from the population, wherein the first gene of interest has been loaded onto the autonomously replicating synthetic chromosome and the recyclable marker gene cassette is no longer present; in the identified first group of cells, using the first recombination system to integrate a second delivery vector comprising the recyclable marker gene cassette and a second gene of interest into a second of the multiple recombination target sites on the autonomously replicating synthetic chromosome; with the second recombination system using signal sites beta, excising the recyclable marker gene cassette from the autonomously replicating synthetic chromosome, wherein the second gene of interest remains on the autonomously replicating synthetic chromosome; identifying a second group of cells in which the first and the second genes of interest have been loaded onto the autonomously replicating synthetic chromosome and the recyclable marker gene cassette is no longer present; in the identified second group of cells, using the first recombination system to integrate a third delivery vector comprising the recyclable marker gene cassette and a third gene of interest into a third of the multiple site specific recombination target sites on the autonomously replicating synthetic chromosome; and with the second recombination system using signal sites gamma, excising the recyclable marker gene cassette from the autonomously replicating synthetic chromosome, wherein the third gene of interest remains on the autonomously replicating synthetic chromosome. ### Claim 13 (depends on 12) The method of claim 12, wherein the synthetic chromosome is a human synthetic chromosome. ### Claim 14 (depends on 12) The method of claim 12, wherein the first recombination system is a Lambda integrase system. ### Claim 15 (depends on 12) The method of claim 12, wherein the second recombination system is a Cre/lox recombination system. ### Claim 16 (depends on 12) The method of claim 12, wherein the signal sites alpha, beta, and gamma are selected from Lox2272, Lox5171, LoxM2 and LoxP, and each of signal sites alpha, beta, and gamma are distinct. ### Claim 17 (depends on 12) The method of claim 12, wherein the excising steps are performed by introduction of a nucleic acid vector encoding a second recombinase to the cell, introducing a second recombinase protein to the cell, or by delivering an encapsulated second recombinase to the cell. ### Claim 18 (depends on 17) The method of claim 17, wherein the excising steps are performed by introduction of a nucleic acid vector encoding the second recombinase to the cell. ### Claim 19 (depends on 12) The method of claim 12, further comprising determining whether the marker gene has been excised from the autonomously replicating synthetic chromosome by FACS sorting the cell after each excising step. ### Claim 20 (depends on 12) The method of claim 12, further comprising the steps of: identifying a third group of cells in which the first, the second and the third genes of interest have been loaded onto the autonomously replicating synthetic chromosome and the recyclable marker gene cassette is no longer present; in the identified third group of cells, using the first recombination system to integrate a fourth delivery vector comprising the recyclable marker gene cassette and a fourth gene of interest into a fourth of the multiple recombination target sites on the autonomously replicating synthetic chromosome; and with the second recombination system using signal sites delta, excising the recyclable marker gene cassette from the autonomously replicating synthetic chromosome, wherein the fourth gene of interest remains on the autonomously replicating synthetic chromosome. ### Claim 21 (depends on 12) The method of claim 12, wherein each gene of interest is tagged with a different fluorescent marker.