Multiplexed deterministic assembly of dna libraries

Abstract

The present disclosure relates to methods of joining three or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro or in vivo. The method allows the joining of a large number of DNA fragments, in a deterministic fashion. It can be used to rapidly generate nucleic acid libraries that can be subsequently used in a variety of applications that include, for example, genome editing and pathway assembly. Kits for performing the method are also disclosed.

1 .- 28 . (canceled) 
     
     
         29 . A method for generating libraries of polynucleotides, the method comprising:
 (a) amplifying via polymerase chain reaction (PCR), a first pool of polynucleotides, wherein the first pool contains pairs of polynucleotides, wherein each pair contains a first polynucleotide and a second polynucleotide, and wherein each first polynucleotide and each second polynucleotide in a pair comprises a 5′ end and a 3′ end, wherein the amplifying introduces a common overlap sequence comprising one or more recognition sequences for one or more site-specific nucleases onto the 5′ end of a first polynucleotide and the 3′end of a second polynucleotide in a pair from the first pool;   (b) assembling each pair of first polynucleotides and second polynucleotides from the first pool into a single nucleic acid fragment by utilizing the common overlap sequence, wherein the single nucleic fragment for each pair comprises a first polynucleotide and second polynucleotide separated by the common overlap sequence from the 5′ end of the first polynucleotide and the 3′ end of the second polynucleotide, and wherein the 3′ end of the first polynucleotide and the 5′ end of the second polynucleotide in the single nucleic fragment for each pair are located on opposing terminal ends of the single nucleic acid fragment, distal to the one or more site-specific nuclease recognition sequence(s);   (c) combining the single nucleic acid fragments for each pair with a second pool containing insert polynucleotides, wherein each insert polynucleotide in the second pool comprises a first assembly overlap sequence at its 5′ end that is complementary to the 3′ end of the first polynucleotide present within the single nucleic acid fragment and a second assembly overlap sequence at its opposing 3′end that is complementary to the 5′ end of the second polynucleotide present within the single nucleic acid fragment;   (d) assembling the first pool and the second pool into a third pool of circularized products, wherein the assembling is performed via in vitro or in vivo overlap assembly methods, and wherein each circularized product in the third pool comprises an insert sequence from the second pool and a pair of first polynucleotides and second polynucleotides from the first pool;   (e) linearizing each circularized product in the third pool via digestion by one or more site-specific nuclease(s) that recognize(s) the one or more site-specific nuclease recognition sequence(s) located between the first polynucleotide sequence and the second polynucleotide sequence in each of the circularized products in the third pool; and   (f) assembling the linearized products into cloning vectors by in vivo or in vivo cloning methods.   
     
     
         30 . The method of  claim 29 , wherein the first pool is generated by selecting pairs of polynucleotide sequences from a larger set of such sequences such that no polynucleotide from the first pool shares common sequence with any other polynucleotide from the first pool beyond a specified threshold, excluding designed assembly overlap sequences between the pairs of polynucleotides of the first pool and the insert polynucleotides of the second pool, or the pairs of polynucleotides of the first pool and the cloning vector. 
     
     
         31 . The method of  claim 30 , wherein the specified threshold is between 5 and 15 contiguous nucleotides. 
     
     
         32 . The method of  claim 29 , wherein the one or more site-specific nuclease recognition sequence(s) located between the first polynucleotide sequence and the second polynucleotide sequence is a homing nuclease recognition sequence and the one or more site-specific nuclease(s) is a homing endonuclease. 
     
     
         33 . The method of  claim 29 , wherein the common overlap sequence comprises an assembly overlap sequence of at least 1 nucleotide and the assembly in step (b) is performed by an overlap-based DNA assembly method. 
     
     
         34 . The method of  claim 33 , wherein the overlap-based DNA assembly method is selected from splicing and overlap-extension PCR (SOI-PCR) or an in vitro overlap-assembly method. 
     
     
         35 . The method of  claim 34 , wherein the one or more site-specific nuclease recognition sequence(s) present in the common overlap sequence on the 5′ end of the first polynucleotide is complementary to the one or more site-specific nuclease recognition sequence(s) present in the common overlap sequence on the 3′ end of the second polynucleotide in each pair, and wherein the utilizing the common overlap sequences of the first and second polynucleotides in each pair in step (b) entails performing SOE-PCR. 
     
     
         36 . The method of  claim 29 , wherein the utilizing the common overlap sequences of the first and second polynucleotides in each pair in step (b) entails digesting the one or more site-specific nuclease recognition sequences present in the common overlap sequence on the 5′ end of the first polynucleotide and the 3′ end of the second polynucleotide in each pair with one or more site specific nucleases for the one or more site-specific nuclease recognition sequences to generate single-stranded overhangs on the 5′ end of the first polynucleotide and the 3′ end of the second polynucleotide in each pair that comprise complementary sequence; and ligating the complementary sequence present on the single-stranded overhang on the 5′ end of the first polynucleotide and the 3′ end of the second polynucleotide in each pair. 
     
     
         37 . The method of  claim 29 , wherein the assembling of step (d) is performed using an overlap-based DNA assembly method. 
     
     
         38 . The method of  claim 29 , wherein the 3′ end of the first polynucleotide and the 5′ end of the second polynucleotide in the single nucleic acid fragment in each pair comprise an additional set of one or more site-specific nuclease recognition sequences and the first assembly overlap sequence and the second assembly overlap sequence in each insert polynucleotide in the second pool comprise one or more site-specific nuclease recognition sequences. 
     
     
         39 . The method of  claim 38 , wherein the assembling in step (d) entails digesting the additional one or more site-specific nuclease recognition sequences present on the 3′ end of the first polynucleotide and the 5′ end of the second polynucleotide in the single nucleic acid fragment in each pair and the one or more site-specific nuclease recognition sequences present in the first and second assembly sequences in each insert polynucleotide from the second pool with one or more site specific nucleases for the additional one or more site-specific nuclease recognition sequences on the 3′ end of the first polynucleotide and the 5′ end of the second polynucleotide in the single nucleic acid fragment in each pair and the one or more site-specific nuclease recognition sequences present in the first and second assembly sequences in each insert polynucleotide from the second pool to generate a single-stranded overhang on the 3′ end of the first polynucleotide that comprises sequence complementary to sequence present on a single-stranded overhang on the 5′end of the first assembly sequence of an insert polynucleotide from the second pool and a single stranded overhang on the 5′ end of the second polynucleotide that comprises sequence complementary to a sequence present on a single-stranded overhang on the 3′end of the second assembly sequence of the same insert polynucleotide from the second pool; and ligating the complementary sequence present on the single-stranded overhangs. 
     
     
         40 . The method of  claim 29 , wherein the cloning vectors of step (f) comprise one or more site-specific nuclease recognition sequences. 
     
     
         41 . The method of  claim 40 , wherein the assembling in step (1) entails digesting the one or more site-specific nuclease recognition sequences in the cloning vectors with the one or more site-specific nucleases for the one or more site-specific nuclease recognition sequences recognition sequences present in the cloning vectors, wherein the digesting generates single-stranded overhangs on opposing ends of the cloning vectors, wherein the single-stranded overhang on one of the opposing ends of the cloning vector comprises sequence complementary to an end of the linearized product generated in step (e) and the single-stranded overhang on the other of the opposing ends of the cloning vectors comprises sequence complementary to an opposing end of the linearized product generated in step (e); and ligating the complementary sequences present on the single-stranded overhangs of the cloning vectors and the linearized products from step (e). 
     
     
         42 . The method of  claim 29 , wherein the first assembly overlap sequence and the second assembly overlap sequence on each insert polynucleotide in the second pool comprises 1 or more nucleotides that are complementary to the opposing terminal ends of the single nucleic acid fragment. 
     
     
         43 . The method of  claim 29 , wherein, for each pair in the first pool, the first polynucleotide and the second polynucleotide comprises sequence corresponding to a target genomic locus in a host cell. 
     
     
         44 . The method of  claim 29 , wherein each insert polynucleotide in the second pool comprises one or more payload sequences located between the first assembly overlap sequence and the second assembly overlap sequence. 
     
     
         45 . The method of  claim 44 , wherein the one or more payload sequences are selected from promoters, genes, regulatory sequences, nucleic acid sequence encoding degrons, nucleic acid sequence encoding solubility tags; terminators, unique identifier sequence or portions thereof. 
     
     
         46 . The method of  claim 43 , wherein, for each pair in the first pool, the first polynucleotide and the second polynucleotide comprises sequence corresponding to a different target genomic locus in a host cell as compared to each other pair in the first pool. 
     
     
         47 . The method of  claim 44 , wherein each payload sequence in the insert polynucleotides in the second pool is different from the payload sequence in each other insert polynucleotide in the second pool. 
     
     
         48 . The method of  claim 44 , wherein each insert polynucleotide in the second pool is generated by:
 (i) performing PCR on a mixture comprising the payload sequence, a forward primer and a reverse primer, wherein the forward primer comprises from 5′ to 3′, a short stretch of one or more nucleotides complementary to the payload sequence, the first assembly overlap sequence, one or more recognition sequences for one or more site-specific nucleases, the second assembly overlap sequence and a second stretch of one or more nucleotides complementary to the payload sequence and wherein the reverse primer comprises sequence complementary to the payload sequence or to other sequence downstream of the payload sequence, wherein the PCR generates a PCR product comprising from 5′ to 3′, the short stretch of nucleic acid complementary to the payload sequence, the first assembly overlap sequence, the one or more site-specific nuclease recognition sequence(s), the second assembly overlap sequence and the payload sequence;   (ii) circularizing the PCR product via an assembly method selected from the group consisting of SOE-PCR, restriction-ligation, blunt-end ligation, overlap based assembly method and recombination-based method, or any other enzymatic or chemical method of joining two DNA molecules; and   (iii) linearizing the circularized PCR product with one or more site-specific nuclease(s) that recognize the one or more site-specific nuclease recognition sequence(s), thereby generating the second pool of polynucleotides.