Cell-free production of ribonucleic acid

Abstract

Provided herein, in some aspects, are methods and compositions for cell-free production of ribonucleic acid.

1 . A method of producing a cell lysate for cell-free production of ribonucleic acid (RNA), the method comprising:
 (a) culturing cells to a desired cell density, wherein the cells comprise at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding a nuclease that includes a protease-recognition site and is linked to a periplasmic-targeting sequence, wherein at least one endogenous ribonuclease is genetically inactivated or inactivated via targeted proteolysis in the cells;   (b) lysing cells produced in step (a), thereby producing a first cell lysate, and   (c) incubating the first cell lysate under conditions that result in RNA depolymerization, thereby producing a first cell lysate containing nucleotide 5′-monophosphates.   
     
     
         2 . The method of  claim 1 , wherein the promoter operably linked to a sequence encoding a nuclease is inducible. 
     
     
         3 . The method of  claim 1  further comprising:
 (d) culturing cells that comprise (i) at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding a cognate protease that cleaves the protease-recognition site of the nuclease, wherein the cognate protease is linked to a periplasmic-targeting sequence, (ii) at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding a nucleotide kinase, (iii) at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding an RNA polymerase, and (iv) an engineered deoxyribonucleic acid (DNA) template containing a promoter operably linked to a sequence encoding an RNA, wherein at least one endogenous ribonuclease is genetically inactivated or inactivated via targeted proteolysis in the cells; and 
 (e) lysing cultured cells produced in step (d), to produce a second cell lysate. 
 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the nuclease is selected from the group consisting of S1 Nuclease, NucA, PNPase, RNase II, RNase III, and RNase R. 
     
     
         6 . The method of  claim 1 , wherein the cells contain at least one protease-targeted endogenous ribonuclease and/or at least one chromosomal deletion in a gene encoding an endogenous ribonuclease. 
     
     
         7 . The method of  claim 6 , wherein at least one endogenous ribonuclease is selected from the group consisting of RNase III, RNase I, RNaseR, PNPase, RNase II, RNase T, RNase E and combinations thereof. 
     
     
         8 . The method of  claim 3 , wherein the nucleotide kinase is a nucleotide monophosphate kinase or a nucleotide diphosphate kinase. 
     
     
         9 . The method of  claim 8 , wherein the nucleotide monophosphate kinase is selected from the group consisting of uridylate kinase, cytidylate kinase, guanylate kinase, and adenylate kinase, and/or the nucleotide diphosphate kinase is selected from the group consisting of nucleoside phosphate kinase, pyruvate kinase, and polyphosphate kinase. 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein at least one endogenous enzyme that degrades polyphosphate is genetically inactivated in the cells. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 12 , wherein the cells contain at least one protease-targeted enzyme that degrades polyphosphate and/or at least one chromosomal deletion in a gene encoding enzyme that degrades polyphosphate. 
     
     
         15 .- 18 . (canceled) 
     
     
         19 . The method of  claim 3 , wherein the cells of step (d) comprise at least two engineered nucleic acids, each containing a promoter operably linked to a sequence encoding an RNA polymerase, wherein one of the engineered nucleic acids encodes a DNA-dependent RNA polymerase, and one of the engineered nucleic acids encodes an RNA-dependent RNA polymerase. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The method of  claim 3 , wherein the engineered DNA template is located on an expression vector containing an endonuclease cleavage site. 
     
     
         24 . The method of  claim 23 , wherein the endonuclease cleavage site is an I-SceI endonuclease cleavage site 
     
     
         25 - 29 . (canceled) 
     
     
         30 . The method of  claim 3  further comprising:
 combining the first cell lysate produced in step (b), the second cell lysate produced in step (e), and polyphosphate, thereby producing a mixture; and 
 incubating the mixture under conditions that result in production of RNA. 
 
     
     
         31 . The method of  claim 1 , wherein the RNA is double-stranded RNA (dsRNA). 
     
     
         32 . A method of producing a cell lysate for cell-free production of ribonucleic acid (RNA), the method comprising:
 (a) culturing cells to a desired cell density, wherein the cells comprise (i) at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding a nuclease that includes a protease-recognition site and is linked to a periplasmic-targeting sequence, and (ii) at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding a nucleotide kinase, wherein at least one endogenous ribonuclease is genetically inactivated or inactivated via targeted proteolysis in the cells;   (b) lysing cells produced in step (a), thereby producing a first cell lysate, and   (c) incubating the first cell lysate in the presence of polyphosphate under conditions that result in RNA depolymerization, thereby producing a first cell lysate containing a mixture of nucleotide 5′-triphosphates.   
     
     
         33 . The method of  claim 32  further comprising:
 (d) culturing cells that comprise (i) at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding a cognate protease that cleaves the protease-recognition sequence of the nuclease, wherein the cognate protease is linked to a periplasmic-targeting sequence, (ii) at least one engineered nucleic acid containing a promoter operably linked to a sequence encoding an RNA polymerase, and (iii) an engineered deoxyribonucleic acid (DNA) template containing a promoter operably linked to a sequence encoding a messenger RNA (mRNA) that includes complementary domains linked by a hinged domain, wherein at least one endogenous ribonuclease is genetically inactivated or inactivated via targeted proteolysis in the cells; and 
 (e) lysing cultured cells produced in step (d), to produce a second cell lysate. 
 
     
     
         34 . The method of  claim 33  further comprising:
 combining the first cell lysate produced in step (b), the second cell lysate produced in step (e), and optionally polyphosphate, thereby producing a mixture; and 
 incubating the mixture under conditions that result in production of RNA. 
 
     
     
         35 . (canceled) 
     
     
         36 . A cell lysate produced by the method of  claim 1 . 
     
     
         37 - 40 . (canceled) 
     
     
         41 . A cell-free method of producing ribonucleic acid (RNA), the method comprising:
 (a) combining a first cell lysate with a second cell lysate, wherein
 the first cell lysate comprises (i) a nuclease that includes a protease-recognition site, and (ii) nucleotide 5′-monophosphates, and 
 the second cell lysate comprises (iii) a cognate protease that cleaves the protease-recognition site of the nuclease, (iv) nucleotide kinase, (v) an RNA polymerase, and (vi) an engineered deoxyribonucleic acid (DNA) template containing a promoter operably linked to a sequence encoding an RNA of interest, thereby forming a reaction mixture; and 
   (b) incubating the reaction mixture under conditions that result in production of the RNA of interest.   
     
     
         42 .- 45 . (canceled)