DNA vector production system

Abstract

A vector production system is provided. The system comprises recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter and the cells include an expression vector encoding a nucleic acid of interest within the regulatory elements of the expression vector which are flanked on either side by a target sequence for at least the first recombinase. The vector production system provides an efficient one-step process for producing linear or circular covalently closed vectors that incorporate a nucleic acid sequence of interest.

We claim: 
     
       1. A vector production system comprising recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter, wherein said cells comprise an expression vector adapted to produce a bacterial sequence-free vector, said expression vector comprising an e xpression cassette, and a nucleic acid of interest flanked on either side by a target binding sequence for the Tel recombinase and integrated within non-binding regions of the Tel target binding sequence are target binding sequences for one or more additional recombinases, wherein the nucleic acid of interest encodes a nuclease genome editing system. 
     
     
       2. The system of  claim 1 , wherein the inducible promoter is thermally-regulated, the IPTG regulated, glucose-regulated, T7 polymerase regulated, cold-shock inducible, pH inducible, or combinations thereof. 
     
     
       3. The system of  claim 1 , wherein the vector comprises the target binding sequence for one or more additional recombinases selected from the group consisting of the telRL site, the loxP site, φpK02 telRL site, the FRT site, phiC31 attP site and the λ attP site. 
     
     
       4. The system of  claim 3 , wherein the vector comprises each of said target binding sequences. 
     
     
       5. The system of  claim 3 , wherein the vector comprises the Tel recombinase pal site and the telRL, loxP and FRT recombinase target binding sequences integrated within the pal site. 
     
     
       6. The system of  claim 1 , wherein the recombinase is selected from telN and tel, the vector incorporates the target binding sequence for at least said recombinase and said system produces a linear covalently closed vector. 
     
     
       7. The system of  claim 1 , wherein the recombinase is selected from cre and flp, the vector incorporates the target binding sequence for at least said recombinase and said system produces a circular covalently closed vector. 
     
     
       8. An expression vector adapted to produce a bacterial sequence-free vector, said expression vector comprising an expression cassette, and a nucleic acid of interest flanked on either side by a target binding sequence for the Tel recombinase and integrated within non-binding regions of the Tel target binding sequence are target binding sequences for one or more additional recombinases, wherein the nucleic acid of interest encodes a nuclease genome editing system. 
     
     
       9. The vector of  claim 8 , wherein the one or more additional target binding sequences are selected from the group consisting of telRL site, the loxP site, □K02 telRL site, the FRT site, phiC31 attP site and the λ attP site. 
     
     
       10. The vector of  claim 9 , comprising each of said target binding sequences. 
     
     
       11. The vector of  claim 8 , comprising the Tel recombinase pal site and telRL, loxP and FRT recombinase target binding sequences integrated within the pal site. 
     
     
       12. A method of producing a linear or circular covalently closed vector comprising incubating a vector production system as defined in  claim 1  under conditions suitable to permit expression of the first recombinase to result in a linear or circular covalently closed vector. 
     
     
       13. The system of  claim 1 , wherein the Tel recombinase target binding site is the phage PY54 Tel 142 base pair target binding site. 
     
     
       14. The system of  claim 1 , wherein the nuclease genome editing system is selected from the group consisting of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease system, a Transcription Activator-Like Effector Nuclease (TALEN) and a Zinc-Finger Nuclease (ZFN). 
     
     
       15. The system of  claim 14 , wherein the nuclease genome editing system is a CRISPR nuclease system comprising a Cas9 nuclease. 
     
     
       16. The vector of  claim 8 , wherein the nuclease genome editing system is selected from the group consisting of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease system, a Transcription Activator-Like Effector Nuclease (TALEN) and a Zinc-Finger Nuclease (ZFN). 
     
     
       17. The vector of  claim 16 , wherein the nuclease genome editing system is a CRISPR nuclease system comprising a Cas9 nuclease. 
     
     
       18. A vector production system comprising recombinant cells designed to encode at least a first recombinase under the control of an inducible promoter, wherein said cells comprise an expression vector adapted to produce a bacterial sequence-free vector, said expression vector comprising backbone sequence, an expression cassette, and a nucleic acid of interest flanked on either side by a target binding sequence for the Tel recombinase and integrated within non-binding regions of the Tel target binding sequence are target binding sequences for one or more additional recombinases, wherein the cells are genetically modified to encode a nuclease genome editing system adapted to cleave the expression vector at a site within the backbone sequence. 
     
     
       19. The vector production system as defined in  claim 18 , wherein the nuclease genome editing system is a CRISPR nuclease system comprising a Cas9 nuclease and gRNA, and the expression vector comprises a target sequence for the gRNA within the backbone sequence. 
     
     
       20. A method of producing a bacterial sequence-free vector having covalently closed ends comprising:
 incubating a vector production system comprising recombinant cells designed to encode at least a first recombinase under control of an inducible promoter and to encode a nuclease genome editing system, under suitable conditions for expression of the first recombinase and the nuclease genome editing system,   wherein the cells comprise an expression vector for producing the bacterial sequence-free vector having covalently closed ends, the expression vector comprising:   a backbone sequence containing a cleavage site for the nuclease genome editing system,   an expression cassette comprising a nucleic acid sequence of interest,   a target sequence for the first recombinase flanking each side of the expression cassette, and   one or more additional target sequences for one or more additional recombinases integrated within non-binding regions of the target sequence for the first recombinase.   
     
     
       21. The method of claim 20, further comprising harvesting the bacterial sequence-free vector. 
     
     
       22. The method of claim 20, wherein the inducible promoter is thermally-regulated, chemically-regulated, IPTG regulated, glucose-regulated, arabinose inducible, T7 polymerase regulated, cold-shock inducible, pH inducible, or a combination thereof. 
     
     
       23. The method of claim 20, wherein the target sequence for the first recombinase and the one or more additional target sequences for the one or more additional recombinases are selected from the group consisting of the PY54 pal site, the N15 telRL site, the loxP site, the φK02 telRL site, the FRT site, the phiC31 attP site, and the λ attP site. 
     
     
       24. The method of claim 23, wherein the expression vector comprises each of the target sequences. 
     
     
       25. The method of claim 23, wherein the expression vector comprises the Tel recombinase pal site and the telRL, loxP, and FRT recombinase target sequences integrated within the pal site. 
     
     
       26. The method of claim 20, wherein the bacterial sequence-free vector is a linear covalently closed vector. 
     
     
       27. The method of claim 26, wherein the first recombinase is selected from telN and tel. 
     
     
       28. The method of claim 20, wherein the bacterial sequence-free vector is a circular covalently closed vector. 
     
     
       29. The method of claim 28, wherein the first recombinase is selected from cre and flp. 
     
     
       30. The method of claim 20, wherein the target sequence for the first recombinase is the phage PY54 Tel 142 base pair target site. 
     
     
       31. The method of claim 20, wherein the nuclease genome editing system is selected from the group consisting of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease system, a Transcription Activator-Like Effector Nuclease (TALEN), and a Zinc-Finger Nuclease (ZFN). 
     
     
       32. The method of claim 31, wherein the nuclease genome editing system is a CRISPR nuclease system comprising a Cas nuclease. 
     
     
       33. The method of claim 20, wherein the nucleic acid sequence of interest encodes a nuclease genome editing system that is selected from the group consisting of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease system, a Transcription Activator-Like Effector Nuclease (TALEN), and a Zinc-Finger Nuclease (ZFN). 
     
     
       34. The method of claim 33, wherein the nuclease genome editing system is a CRISPR nuclease system comprising a Cas nuclease. 
     
     
       35. The method of claim 20, wherein the expression vector further comprises at least one enhancer sequence flanking each side of the target sequence for the first recombinase to facilitate nuclear uptake and enhance transfection efficiency of the bacterial sequence-free vector and expression of the nucleic acid sequence of interest. 
     
     
       36. The method of claim 35, wherein the at least one enhancer sequence is a SV40 enhancer sequence. 
     
     
       37. The method of claim 35, wherein the system comprises at least two enhancer sequences flanking each side of the target sequence for the first recombinase. 
     
     
       38. The method of claim 37, wherein the at least two enhancer sequences are SV40 enhancer sequences. 
     
     
       39. The method of claim 20, wherein the target sequence for the first recombinase and the one or more additional target sequences for the one or more additional recombinases have a total base pair length of less than or equal to 142 base pairs. 
     
     
       40. The method of claim 20, wherein the one or more additional target sequences for the one or more additional recombinases is at least five additional target sequences for at least five additional recombinases. 
     
     
       41. The method of claim 40, wherein the target sequence for the first recombinase and the at least five target sequences for the at least five additional recombinases have a total base pair length of less than or equal to 300 base pairs.