SYSTEM AND METHOD FOR EDITING AND MANIPULATING DNA

FIELD OF THE INVENTION The present invention relates to a system and a method for manipulating, processing and editing DNA molecules, and in particular, to such a system and method in which a protocol for synthesizing target DNA molecule is abstracted utilizing a core operation on a given input DNA molecule.

BACKGROUND OF THE INVENTION

Composition and manipulation of DNA molecules are important tasks for molecular biology in both research and industrial applications. While the problem of de novo DNA composition has been addressed systematically (5, 10-12, 16, 17), no general method for editing DNA molecules is available, and specific editing tasks are presently addressed by specialized labor-intensive methods such as site- directed mutagenesis (1 -3) and/or methods relying on the use of restriction enzymes (6, 7).

Biology labs engage daily in manual labor-intensive DNA processing — the creation of variations and combinations of existing DNA — using a plethora of methods such as site-directed mutagenesis( 1,2,3), error-prone PCR(4), assembly PCR(5), cleavage and ligation(6,7), homologous recombination(8,9), and others(10,l 1, 12, 13, 14, 15). So far no uniform method for DNA processing has been proposed and, consequently, no engineering discipline has been able to eliminate the manual labor associated with DNA processing. DNA composition, also called de novo DNA synthesis, is typically achieved by assembling synthetic oligonucleotides into ever longer pieces using one of several methods ( 17, 18). Although much progress has been made in achieving uniform, efficient and automated methods for performing de novo DNA synthesis ,for example by avoiding cloning steps (17), while others use DNA microchips to reduce costs and errors ( 18) which occur very frequently in these de novo synthesis methods, still these methods suffer from many drawbacks. DNA editing, on the other hand, has no systematic solution to date, and the various editing tasks are performed by a plethora of labor-intensive methods (1 -3). Site- directed mutagenesis generates targeted changes including single or multiple

nucleotide insertions, deletions or substitutions, generally via the use of an oligonucleotide primer that introduces the desired modification. These fall into two major categories: those based on primer extension on a plasmid template (1 -3), and PCR-based methods (13). Other methods use a restriction enzyme to cut the DNA molecule at specific preplanned sites of specific sequence and enable the ligation of a DNA fragment that contains the matching sites on its ends. In this method the short restriction sites must be specific and unique in the sequence to avoid undesired restrictions. Other methods generate random mutations using error-prone PCR, incremental truncation or random DNA shuffling. DNA shuffling performs in vitro homologous recombination of pools of selected homological DNA fragments by random fragmentation and PCR reassembly. However, this methodology is inefficient if multiple non-random sequence manipulations are required, as it requires iterative stages of mutagenesis, cloning, sequencing and selection. Therefore, current DNA processing methods do not provide for seamless DNA editing and manipulations that allow for the abstraction and creation of custom made, designer DNA molecules.

SUMMARY OF THE INVENTION There is an unmet need for, and it would be highly useful to have, a system and a method for seamless manipulating and editing of existing DNA strands for the purpose of creating customized and/or designer DNA strands. The background art does not teach or suggest a DNA editor, which enables both composition and editing of DNA strands. The present invention overcomes the drawbacks of the background art by providing a system and method for DNA editing based on a core operation. The core operation according to a preferred embodiment of the present invention enables the system and method of the present invention by providing for seamless DNA editing and manipulation preferably by optimizing the number of core operations required to create the targeted DNA strand.

Representation of a DNA molecule is accomplished by the use of a continuing sequence using a 4 letter code. Each letter of the 4 letter code represents a different nucleic acid found in a DNA strand, namely, A for adenine, T for thymine, C for cytosine and G for Guanine. Therefore, DNA strands, no matter

how long may be represented in a textual manner using a running sequence of the 4 letter code. Although a text editor such as MS Word allows composing new text and editing an existing piece of text within the same framework, no such DNA composition, apparatus or system has been made available that allows one to manipulate, edit, visualize or otherwise electronically manipulate DNA strands. Moreover, the background art does not teach or suggest a system or method for the automated production of DNA based on its sequence representation for longer strands. Proof of concept systems have demonstrated the construction of 15kbp DNA fragments (18), yet such systems still suffer from many drawbacks, including difficulty of use, expense and the potential for introduction of errors.

Therefore an efficient DNA editing method, such as that provided by the present invention, enables extensive manipulation of a DNA molecule while maximizing the use of existing DNA components, similarly to the way a text editor enables efficient editing of an existing text using operation such as insert, delete, substitute, cut, copy and paste. However, no such system, device or method has been offered or made apparent in the background art. The present invention overcomes these drawbacks of the background art and in addition, in at least some embodiments, preferably provides an error free or at least a substantially error free method for such editing, wherein "substantially error free" refers to an error rate of at least less than about 5%, preferably less than about 3%, more preferably less than about 2% and most preferably less than about 1%.

Within the context of this application the term "core operation", "core function", "Y operation", "core Y operation", "Y function", "Y", may be used interchangeably to refer to a process wherein DNA is manipulated, preferably wherein two DNA fragments are concatenated into a single DNA molecule.

Within the context of this application the term editor, DNA editor, DNA processor, DNA manipulation, interchangeably refer to a system and method for editing DNA molecule according to a preferred embodiment of the present invention. Within the context of this application the term edit, editing, processing or the like collectively refers to at least one or more process, step, reaction or the like measure taken to manipulate DNA for example including but not limited to at least one or more cut, copy, paste, insert, delete, replace, substitute, cut and paste, copy and paste, taken alone or in any combination thereof.

Within the context of this application the term "target", "target molecule", target DNA strand, refers to a DNA molecule that is created by an optional operation of editing an existing DNA molecule according to an optional embodiment of the system and method of the present invention. Within the context of this application the term "input", "input fragment",

"input molecule", "input DNA strand", "input DNA fragment" may be interchangeably used in referring to at least one or more initial DNA molecules used by the system and method of the present invention to create a targeted DNA molecule. Within the context of this application the terms "DNA fragment", "DNA strand", "DNA molecule", "DNA sequence", may be used interchangeably to refers to an oligonucleic acid sequence.

Within the context of this application the term "division point" refers to any point within a DNA fragment that may be potentially useful for implementing a core Y operation. Preferably, a division point defines a point within a DNA fragment from which at least two, a left and right, flanking sub-sequences may be defined. Most preferably, a core Y operation is utilized to concatenate two different flanking sub-sequences.

According to a preferred embodiment of the present invention, the present invention provides for DNA manipulation and/or editing through the use of a core operation interchangeably referred to a "Y" operation. Most preferably, the Y

„. operation provides for DNA manipulation by joining two double stranded DNA fragments into one double stranded DNA molecule. The core operation according to the present invention may be applied to a plurality of DNA molecules, and is preferably independent of the DNA sequence itself therefore not limiting the core operation to particular DNA sequence.

Preferably, the core operation is independent of the DNA sequence size and may be applied to DNA molecules of varying size. Most preferably, a DNA molecule undergoing a Y operation is amenable to amplification, also most preferably by PCR. Optionally, other amplification techniques as may be implemented with the Y operation according to the present invention. Amplification with PCR provides for a Y operation to be carried out on a DNA molecule of varying size optionally up to 6kbp, optionally up to 5kbp, preferably up to 4kbp, optionally and preferably up to 3kbp, and most preferably from about 100 bp to

about 4kbp. Optionally and preferably the DNA molecule's size limitation is depicted by the amplification technique utilized.

The core function according to the present invention may optionally and preferably be applied in at least one or more sequences, for example including but not limited to a stepwise manner, in series, in parallel, in sequence, in a loop, or in any repeated manner to provide for DNA editing functions. DNA editing functions may for example include but are not limited to at least one or more of cut, copy, paste, insert, delete, replace, substitute, cut and paste, copy and paste, taken alone or in any combination. Most preferably, a plurality of core operations may be combined together to form a protocol for editing, abstracting, customizing DNA strands.

Among the advantages of the core Y operation, over background art methods, is the independence from site specific restriction enzymes relating to a particular sequence that is that such primers and overlap regions may be defined based on the target region and therefore may be defined for almost any location on a DNA sequence, unlike the use of restriction enzymes which require that a specific restriction site be uniquely embedded within a target DNA fragment.

Most preferably the primers utilized for individual core operation may be designed and optionally selected from an available DNA library. Most preferably the primers are depicted based on the edit protocol performed by at least one Y operation. Primer selection and/or design is optionally dependent on the amplification technique utilized as described above. Optionally the primer utilized comprises up to about 100 bp, more preferably the primers comprise up to about 80 bp in length. According to a further embodiment, the present invention provides for a method for implementing the core function to produce customized DNA molecules. Preferably, the method according to the present invention utilizes at least one or more input DNA molecules to abstract a protocol applying the core function, according to the present invention, that would produce at least one or more target DNA molecules. Most preferably the abstracted protocol maps a customized targeted DNA molecule that may be composed of at least one or more input DNA molecules by implementing at least one or more core operation according to the present invention. Optionally and preferably, an abstracted protocol may be optimized according to at least one or more controllable factors for example

including but not limited to the number of core operations required, the input molecules used, the number of intermediate steps, the number of de novo sequencing steps or processes required, primer's required, primer length, use of shorter primers, use of longer primers, primer melting point (Tm), or the like controllable factors

According to some embodiments of the present invention, a method for optimizing the abstracted protocol is provided. Preferably, a Divide-and-Conquer

(D&C) DNA editing algorithm is used for determining a protocol of core operations for composing a target DNA molecule from at least one or more available input DNA molecules preferably available in a DNA library.

Most preferably, the method for optimizing and abstracting the protocol comprises: initially defining a target and DNA input library. Next there is preferably a preprocessing phase wherein all potential input DNA fragments, comprising a DNA input library, are evaluated in light of the targeted DNA sequence molecule. Most preferably, the evaluation process provides for the identification of targeted DNA fragment sequences within at least one or more input DNA molecule.

Next, preferably a process is performed to identify and mark all the middle points and end points in the target DNA molecule, defining potential division points based on individual optional DNA input sequences. Next preferably the process involves implementing a recursive protocol planning procedure, which preferably utilizes the divide-and-conquer strategy, wherein each recursive application of the planning procedure, the marked input sequence is divided into two adjacent parts at a selected point defining at least one or more division points. Preferably, a division point is chosen by comparing the target fragment to all input fragments identifying identical subsequences (referred to as NF). Next, the boundaries of each NF are marked.

Next, all potential division points are optionally and preferably considered and scored according to three rules: a. First, each division point earns a score that is proportional to the sum of the sizes of the largest NFs that are wholly found on both sides of the division point, the larger the target the score. Preferably this is to prevent dividing a large NF in two instead of using it as is.

b. Second, each point is given a penalty that is proportional to the distance from the boundary from the closest NF as this causes the use of synthetic fragments and it is preferable to minimize this. c. Third, a point is given a small penalty for the distance from the center of the fragment. Given two identical points the construction tree is preferably balanced.

The points are then sorted from high to low score and are considered sequentially. Once the candidate division points are sorted, the best division point is selected and the algorithm tries to plan a basic stage reaction that will combine the two sub- fragments induced by the division point into the target molecule: a. The necessary primers are planned and validated for specificity, affinity (Tm), dimerization and length constraints for both PCR amplification and Elongation reactions of the basic stage. b. If valid primers are not found the algorithm continues with the next potential division point. c. In the case that a valid division point is found the procedure is called recursively on both the left sub-fragment and the right sub-fragment. d. If a protocol for one of the sub- fragments could not be found the procedure tries the next potential division point, e. If no possible division is found the recursive procedure call returns with failure value, which causes the calling procedure to try the next best division point. The recursive division ends when the input to a recursive call can be extracted from one of the input fragments or when it is small enough to be produced as a synthetic oligo.

As output, the algorithm returns the editing protocol in the form of a binary tree. The leaves are either existing DNA fragments (with valid PCR primers) or synthetic oligos. Each internal node corresponds to a dsDNA intermediate product that can be built using a Y operation from its two sons, which result a simple iterative protocol. The root of the tree is the target molecule T. The output of the algorithm also provides the list of primers needed to execute the protocol.

In the absence of relevant input fragments the system preferably defaults to automatic recursive composition of the target molecule from synthetic oligonucleotides.

A further embodiment of the present invention provides for a system that automatically synthesize and edit DNA molecules preferably comprising a user interface for abstracting the protocol for synthesizing a target DNA fragment from existing DNA molecules, at least one or more input DNA fragments to be edited or combined for creating the target DNA fragment, a DNA synthesizer for synthesizing oligonucleotides for performing the editing steps in producing the target DNA molecule by implementing the core function of the abstracted protocol according to the present invention, a PCR machine for carrying out enzymatic reactions. Optionally and preferably the system according to an optional embodiment may further comprise an automatic sequencer preferably for determining, testing and repairing the produced target sequences

Unless otherwise defined the various embodiment of the present invention may be provided to an end user in a plurality of formats, platforms, and may be outputted to at least one of an assembly line, a robot, a computer readable memory, a computer display device, a printout, a computer on a network or a user. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The materials, methods, and examples provided herein are illustrative only and not intended to be limiting. Implementation of the method and system of the present invention involves performing or completing certain selected tasks or steps manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of preferred embodiments of the method and system of the present invention, several selected steps could be implemented by hardware or by software on any operating system of any firmware or a combination thereof. For example, as hardware, selected steps of the invention could be implemented as a chip or a circuit. As software, selected steps of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In any case, selected steps of the method and system of the

invention could be described as being performed by a data processor, such as a computing platform for executing a plurality of instructions.

Although the present invention is described with regard to a "computer" on a

"computer network", it should be noted that optionally any device featuring a data processor and/or the ability to execute one or more instructions may be described as a computer, including but not limited to a PC (personal computer), a server, a minicomputer, a cellular telephone, a smart phone, a PDA (personal data assistant), a pager. Any two or more of such devices in communication with each other, and/or any computer in communication with any other computer, may optionally comprise a "computer network".

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in order to provide what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings: Figures IA-B are schematic diagrams illustrating exemplary embodiments of the core operation according to the present invention.

Figures 2A-I are exemplary molecular DNA editing functions made possible by implementing the core operation of Figures 1 A- I B.

Figures 3A-B are exemplary optional insert edit function protocols according to the present invention wherein the same input and target molecules are utilized however using a different pathway and intermediates.

Figure 4 is a flow chart of a method according to exemplary embodiments of the present invention for abstracting an editing protocol using core operation.

Figures 5A-B are schematic block diagrams of an exemplary system according to some embodiments of the present invention for producing targeted DNA molecules by implementing the core operation.

Figure 6A provides an exemplary editing protocol map according to the system and method of the present invention in the form of a binary tree, as illustratd in Example 1. Figure 6B provides a schematic diagram of the input and output DNA molecules provided for by the editing protocol binary tree of Figure 6A as non- limiting examples only.

Figure 7A provides schematic diagrams of the input and output DNA molecules provided for by an optional, exemplary editing protocol as depicted in Example 3 as non-limiting examples only.

Figures 7B-D provide schematic diagrams of exemplary editing protocol maps in the form of a binary tree according to some illustrative embodiments of the system and method of the present invention, relating to the input and output sequences of Figure 7A. Figure 8A provides schematic diagrams of the input and output DNA molecules provided for by an optional editing protocol as depicted in Example 4 as non-limiting examples only.

Figure 8B provides a schematic diagram of an exemplary editing protocol map in the form of a binary tree according to some illustrative embodiments of the system and method of the present invention, relating to the input and output sequences of Figure 8A.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a system and a method creating customized targeted DNA molecules from existing input DNA molecules. Targeted DNA sequences are preferably obtained by manipulating and editing exiting DNA molecules therein saving the costs, effort and errors associated with de-novo synthesis of DNA sequences. Most preferably the DNA editor according to the

present invention rely on a core operation that when implemented in various combination on different DNA molecules may bring about more complex editing operation. The present invention relates to a system and a method for manipulating, processing and editing DNA molecules, and in particular, to such a system and method in which a protocol for synthesizing target DNA molecule most preferably from existing input DNA molecules is abstracted utilizing a core operation on a given input DNA molecule.

The principles and operation of the present invention may be better understood with reference to the drawings and the accompanying description. Figure IA provides a schematic diagram of the core operation according to the present invention wherein a first DNA input fragment A is concatenated with a second DNA input fragment B to form a targeted DNA fragment AB, that is preferably and optionally a concatenated DNA molecule of the input DNA fragments. Most preferably, the core Y operation may be utilized to edit DNA in a plurality of ways for example providing for edit operations for example including but not limited to at least one or more cut, copy, paste, insert, delete, replace, substitute, cut and paste, copy and paste, taken alone or in any combination thereof. Preferably, the core Y operation may be implemented on a plurality of DNA forms for example including but not limited to single stranded, double stranded or partially hybridized DNA, or the like.

Optionally, at least one or more of the input fragments may be found within a common DNA fragments. Optionally, at least one or more of the input fragments may be found on individual DNA fragments. Figure I B depicts the method by which the core Y operation depicted in

Figure IA is most preferably implemented. In stage 1 10 DNA fragments A and B form are defined as an input DNA fragment A and B which are to be concatenated. In stage 1 12, DNA input fragments A and B are amplified, preferably using PCR, with primers 1 14 preferably comprising an overhang to produce overlapping dsDNA 1 12. Most preferably primers 1 14 are phosphorylated at their 5' end, most preferably comprising up to about 80bp. Most preferably stage 1 12 produces a phosphorylated double stranded primer extended input DNA molecule 1 16. In stage 1 18 the phosphorylated double stranded primer extended input DNA molecules 1 16 are digested, preferably producing two overlapping single stranded

DNA 120. Preferably, the digestion is undertaken with an exonuclease for example including but not limited to a λ-exonuclease. Most preferably the overlap region enables each of the single stranded DNA 120 to anneal and associate with one another forming complex 122. Most preferably the overlapping region of complex 122 is corresponds to primer 1 14. In stage 124 complex 122 is preferably elongated producing the targeted DNA sequence 124, most preferably a concatenated sequence comprising the input DNA fragments A and B. Most preferably, elongation is achieved with a mutual elongation with a DNA polymerase. Optionally and preferably, the polymerase used in stage 124 is in quasi-equilibrium, wherein the PCR process is amended to provide slow heating and cooling processes preferably improving the energetic efficiency of the reaction.

Figures IA and IB provide a depiction of the core Y operation according to the present invention. However, for the core Y operation to be successful it must be appropriately planned according to a preferred embodiment of the present invention. Most preferably, the core operation must be planned due to chemical constraints that allow for the concatenation of individual input DNA fragments. For example, primer 1 14 and overlap specificity are chemical constraints that must be addressed to ensure the success of the core Y operation. However, the chemical constraints provide the strength and flexibility of the preferred embodiment of the present invention. Among the advantages of the core Y operation, over background art methods, is the targeted approach, allowing it to be independent of limitations associated with background art methods comprising the use of site specific restriction enzymes that must be related to a particular sequence within a targeted DNA molecule. Moreover, the targeted design provided by the system and method of the present invention is such that primers and overlap regions may be defined based on the target region and therefore may be defined for almost any location on a given DNA sequence.

Figure 2A-I depicts a plurality of optional editing functions provided for by the implementation of the core Y operation, for example including but not limited to at least one or more of cut, copy, paste, insert, delete, replace, substitute, cut and paste, copy and paste, taken alone or in any combination thereof. The edit operations depicted in Figures 2A-I are made possible with the implementation of a plurality of performances, in varying combinations, of the basic core Y operation.

Figure 2A depicts the paste operation of wherein input DNA sequences A, B, C and D are concatenated to form the targeted DNA strand ABCD. Optionally, the core operation depicted in Figure IA may be applied in 3 instances to form the targeted sequence 206. Optionally, a first Y operation may be implemented wherein input DNA fragment A is concatenated with input DNA fragment B to form an intermediate DNA fragment AB 202. Similarly a second core Y operation may be implemented wherein input DNA fragment C is concatenated with input DNA fragment D to form an intermediate DNA fragment CD 204. A third core Y operation may be implemented to concatenate intermediate DNA fragments AB 202 and intermediate DNA fragments CD 204 to form the targeted DNA fragment 206. The target DNA molecule 206 may be formed in a plurality of optional combination of using at least one or more core operation. A preferred embodiment of the present invention provides for the identification of the optimized use of the Y operation in creating a protocol map specific to a target DNA molecule based on the available input DNA sequences.

Figure 2B depicts the insert operation wherein a first input DNA sequence C is inserted in a second input DNA sequence AB to form the target DNA molecule ACB 216. Optionally, the core operation depicted in Figure IA may be applied in 2 instances to form the targeted sequence 216. Optionally, a first Y operation may be implemented wherein an input DNA fragment AB is manipulated to delete a subsection B of the input DNA fragment while replacing it by concatenating with input DNA fragment C to form an intermediate DNA fragment AC 212. Therein a single Y operation subsequence B of the input DNA fragment was deleted and replaced by the second input DNA fragment. Similarly a second core Y operation may be implemented wherein input DNA fragment AB is selectively manipulated to delete a subsection A while associating with associated with intermediate DNA fragments AC 212 to form the targeted DNA fragment 216. Figure 2C depicts the delete operation wherein a single Y operation may be utilized to provide a target sequence AC 226 from two copies of a single input sequence ABC. The move from the input sequence ABC to the target sequence AC may be provided with a single Y core operation where proper primer selection and planning could provide to concatenate two subsection namely A and C originating from the same input DNA sequence ABC. The primer selection providing such an edit operation would be specific for the A subsection of the input

molecule and the C subsection of the input molecule. Therein two copies of the input DNA molecule ABC are selectively concatenated to form the target DNA molecule AC 226 therein effectively deleting the B subsection of the input DNA molecule. Figure 2D provides an example of a replace edit function, wherein a particular sequence B within an input sequence of ABC is replaced to form the targeted DNA strand ADC. A first Y operation is performed to produce an intermediate DNA sequence AD that is then associated with the input DNA sequence ABC, however careful primer selection provides for specifically concatenation subsection C of input DNA sequence ABC with the intermediate DNA sequence AD forming the targeted sequence ADC.

Figure 2E depicts an optional protocol to carry out a cut and past edit function utilizing the core operation depicted in Figure IA. For example, an input DNA sequence ABCD may undergo editing to form the target sequence ACBD 246 by cutting the B subsection of the ABCD input sequence and pasting it prior to the D subsection. Optionally, a variety of Y operations may be implemented to synthesize the target molecule from the input molecule. For example, a first Y operation may provide an intermediate DNA strand 242 comprising the sequence equivalent to AC by cutting out both the B sub-sequence and the D sub-sequence of the input molecule. Most preferably this first Y operation is accomplished by utilizing primers that are specific to the A and C sub-sequences. A second Y operation may be implemented to provide intermediate DNA strand 244 comprising the sequence equivalent to BD by cutting out both the A sub-sequence and the C sub-sequence of the input molecule. Most preferably this second Y operation is accomplished by utilizing primers that are specific to the B and D subsequences, effectively joining the two sub- sequences. A third Y operation is implemented to provide the targeted molecule ACBD 246 most preferably by concatenating the two intermediate DNA strands AC 242 and BD 244. Most preferably, concatenation of the two intermediate strands is implemented with a Y operation as described in Figure 2A and I B wherein primers are selected for the overlap region or fusion point wherein subsequence C and B intersect to form the target molecule ACBD 246.

Figure 2F and 2G depict optional protocols to carry out a copy and paste edit function utilizing the core operation depicted in Figure I A both based on the

same input sequence ABCD and forming the target sequence ABCBD. Figure 2F shows input DNA sequence ABCD may undergo editing to form the target sequence ABCBD by copying the B subsection of the input sequence and pasting it prior to the D subsection. Optionally a variety of Y operations may be implemented to synthesize the target molecule from the input molecule. For example, a first Y operation may provide an intermediate DNA strand 252 comprising the sequence equivalent to ABCB by providing a copy of the B subsequence and replacing the D sub-sequence. A second Y operation may be implemented to provide the target molecule ABCBD 256 by utilizing a Y operation between intermediate sequence 252 and the input sequence ABCD 254 to paste the D sub-sequence onto the intermediate sequence 252 forming the target sequence ABCBD 256.

Figure 2G depicts the same input DNA molecule ABCD and target sequences ABCBD, however utilizing a different set of Y operations based on different primers. The core Y operations are utilized to create intermediate sequences ABC 262 and BD 264 that are concatenated in a final Y operation to produce the targeted sequence ABCBD 266. The primers used to obtain the intermediates define the Y operation utilized to bring about the target sequence. For example, complementary primers 263 and 265 provide for the Y operation that links the D sub-sequence with the B subsequence that gives rise to the BD 264 intermediate. Similarly, complementary primers 267 and 269, wherein primer 267 comprises a B sub-sequence overhand with a C sub-sequence, will eventually provide for the Y operation that concatenates the two intermediates 262 and 264 to create the target sequence ABCBD. The difference between Figure 2F and 2G is the protocol utilized to obtain the target sequence from the input sequence. Optionally, either protocol may be used however the method according to a preferred embodiment of the present invention may prefer one or the other depending on at least one or more optimization factors, for example including optimizing the use of available primers, time, number core operations required, use of intermediate products for parallel use, relative size of the fragments that are concatenated or the like optimization factors

Figures 2H and 21 further depict how primer optimization, target sequence and available input molecules determine the number of Y operations required to

perform a the same editing task. For example, Figure 2H depicts a single Y operation required to insert a short DNA sequence C 272 into an input sequence AB 270. Similarly, Figure 21 depicts an insertion edit operation into the same input sequence AB 270 however the DNA sequence C 282 inserted is a larger one. Optionally, for small enough insertion sequences a single Y operation may be utilized, through primer extension, as depicted in Figure 2H. For small sequence insertions the primer utilized is optionally up to about 100 bp and most preferably up to about 80 bp, including the insertion sequence itself. Primer size limitation is dependent on the amplification process utilized for example including but not limited to PCR. Most preferably, the size limitation of the small insert is dependent on the primer utilized, most preferably up to about 80bp. For a larger insertion sequence, as depicted in Figure 21, a plurality of Y operations and a larger number of primers are required to bring about the insert edit function.

Optionally, when implementing small insertion or deletion sequences, for example up to five codon sequences or 15 bases the edit may be implemented through a single Y operation by effectively choosing the appropriate primer related to the insertion or deleted sequence.

Figure 3 A and 3 B further provides a depiction of the two different pathways that may be utilized to obtain the same target DNA sequence from the same input DNA sequence. Optimization preferably provided by the system and method of the present invention could be based on the primers available, intermediate sequences required elsewhere in the process, chemical constraints or the like factors and or variables that may be optimized. For example, in some situation protocol 310 depicted in Figure 3 B may be preferred as it utilized parallel Y operations. While in other situation protocol 300 of Figure 3A would be preferred because of the need for the AEBCD intermediate sequence 302 elsewhere in a parallel process (not shown).

Figure 4 depict an exemplary embodiment of the optimization method according to the present invention where an editing protocol is devised based on a targeted DNA sequences and available input DNA sequences. The method according to an optional embodiment is preferably used to determine which of available editing sequences are to be used for example as shown in Figure 3A and Figure 3 B by optimization at least one or more factors for example including but not limited to available input DNA sequences, available primers, intermediate

sequences required, chemical constraints, sequence of events performed in parallel versus sequential core operations, number of core operations, the amount of input fragments reuse or the like.

In stage 400 at least one or more target DNA sequences and input DNA sequences are preferably defined. Optionally and preferably, available input sequences are chosen from a library or repository of DNA sequences, for example including but not limited to available DNA sequences, primers, enzymes, known genes, commercial sources of DNA, known genomes and the like.

Next in stage 402, a preprocessing phase is performed wherein all input DNA fragments in the target DNA molecule are identified. In stage 404 all input DNA fragments identified in stage 402 are marked preferably at various points along the input sequence for example including but not limited to at least one or more middle points and/or end points that correspond and correlate the input and target DNA sequences. In stage 406, a recursive protocol planning procedure is implemented, preferably utilizing the divide-and-conquer strategy. Preferably, the marked input sequence is divided into two adjacent parts, namely the left sub-sequence and the right sub-sequence, at a selected point defining at least one or more division points. Preferably, a division point is chosen according to at least one or more criteria for example including but not limited to input fragment use, sequence of events performed in parallel versus sequential core operations and balancing the size of the left sub-sequence and the right sub-sequence or the like.

In stage 408 input fragments which are relevant to each sub-sequence or sub-target are identified (referred to as NF) and marking the boundaries of each NF within the context of the subsequence.

In stage 410 all potential division points are considered and scored according to at least three preferred rules. A first rule is optionally wherein division point earns a score that is preferably proportional to the sum of the sizes of the largest NFs that are wholly found on both sides of the division point; more preferably, the larger the NF within the target, the larger the score. This rule prevents dividing a large NF in two instead of using it as whole.

A second rule is preferably implemented to minimize the number of fragments not found within the input DNA fragment library. Each point is given a penalty that is more preferably proportional to the distance from the boundary of the

closest NF. A third rule occurs wherein a division point is given a small penalty, preferably proportional to the distance from the center of the NF. Preferably, the third rule enables a decision to be made between two optional protocols associated with two identical division points. Most preferably the third rule provides for selecting a more preferable and balanced editing protocol tree.

Once scored, individual division points are then sorted in stage 412, most preferably in a descending order based on a score, from high to low score. Each division point is preferably then considered sequentially.

In stage 414, the method according to the present invention most preferably attempts to abstract and/or map at least one or more core Y operation that will combine the two sub-fragments (NF) induced by the division point into the target molecule. Preferably, in stage 414 the necessary primers are planned and validated according to at least one or more factors, for example including but not limited to specificity, affinity, melting temperature Tm, nonspecific dimerization with themselves, nonspecific dimerization with other primers and length constraints. Elongation overlap is also evaluated at this stage including overlap specificity, affinity (Tm), nonspecific dimerization with themselves, nonspecific dimerization with other single stranded fragments and length constraint. Most preferably, primer and overlap validation is performed for both PCR amplification and elongation reactions of the core Y operation. Optionally and preferably, if valid primers are not found the method continues to evaluate the next potential division point, preferably returning to stage 412.

Optionally and preferably, if valid primers are found for a division point, the procedure is called recursively in stage 416 on both the left sub- fragment and the right sub-fragment, preferably returning to stage 408 for each subsequence. Optionally and preferably, if a protocol for at least one of the sub- fragments is not found the procedure attempts the next potential division point, preferably returning to stage 412. Optionally and preferably, if no possible valid division point is found, most preferably the recursive procedure call returns with failure value preferably which causing the calling procedure to try the next best division point.

In stage 418, preferably the recursive division ends when the input to a recursive call can be extracted from one of the input fragments or when it is small enough to be produced in the preferable method of de novo DNA synthesis, preferably cheaply using DNA synthesis machine.

In stage 420 preferably the editing protocol is returned, based upon the output of the above stages, most preferably in the form of a binary tree, as depicted in Figure 5. Optionally, the protocol may be provided in a plurality of formats for example as a set of instructions to an entity including but not limited to a user, a computer, a robot in at least one or more optional formats for example comprising but not limited to an assembly line, a robot, a computer readable memory, a computer display device, a printer, another computer on a network or a user. A further output of the algorithm comprises a list of primers needed to execute the protocol. Figure 5 A depicts an optional embodiment of the present invention with a system 500 that preferably automatically synthesizes and edits DNA molecules from a library of input DNA molecules. System 500 preferably comprises a user interface 502 for abstracting the protocol for synthesizing a target DNA fragment most preferably from an existing library DNA molecules 504, at least one or more input DNA fragments to be edited or combined for creating the target DNA fragment 508, and a DNA synthesizer 506 for synthesizing performing the editing steps in producing the target DNA molecule by implementing the core function of the abstracted protocol according to the present invention. Optionally and preferably, system 500 may further comprise a PCR machine 505 for carrying out the enzymatic reactions. Optionally and preferably system 500 according to an optional embodiment may further comprise an automatic sequencer 507 preferably for determining, testing and repairing the produced target sequences.

As described in the Examples below, the experimental methods were preferably performed by using a laboratory robot. As an illustrative, non-limiting example only, the Tecan Genesis Laboratory Robot can be used. This robot is a modular and programmable open platform. It consists of a modular table space and two robotic arms. The table can be equipped with various carriers and racks for tubes, microplates, tips and reagents, as well as external integrated equipment such as a PCR machine, vacuum manifold, plate readers, etc. One of the robot arms is the liquid handling arm or LIHA, which features 8 disposable-tip pipettes. Each pipette is connected to a different syringe and is thus capable of handling different volumes simultaneously. The LIHA can detect the liquid level in each tube automatically using the robot's disposable tips and can set each pipette to a different height accordingly. A second robot arm is the Robotic Manipulation Arm (ROMA) which

can handle square shaped objects such as microplates, and can load or unload them onto the robot's integrated devices. The robot is controlled by a personal computer (PC) using a software program called Gemini (developed by Tecan Group Ltd.). This program enables the user to run robot scripts called GEM files. The GEM files are in fact files written in the robot assembly language that includes information regarding the robot table organization as well as the script flow. Gemini also supplies the user with a graphic user interface environment for the development of GEM file scripts.

Another role of Gemini is to maintain the system definitions. The definitions include the properties of standard carriers and racks for tubes and microplates and liquid handling policies for various types of liquids and tips. This set of definitions can be extended by an advanced user to integrate new equipment to the robot system.

Actual development of scripts and performance of the experiments was done through the Robolab development environment, shown in more detail in Figure 5B. The Robolab environment includes several processing units, the compiler, the Gemini Software and the Robot.

A Robolab experiment program is written as a text file and is compiled using a compiler called robocom.pl (The compiler is implemented in Perl). The compiler compiles the Robolab experiment program into a GEM file. The GEM file, which is written in the robot assembly language can be executed by the Gemini software, and performs the experiment automatically. The compiler also validates correctness of the program and reports when errors are detected. For this purpose it uses a system configuration file that contains description of the table organization and other pre-defined system information. This enables the compiler to validate that the program may run on the current configured system. Besides the GEM file, the compiler produces a checklist which is used during the actual run-time of the compiled script and is aimed to validate the precondition of the system. In addition, the compiler produces a log file which documents the experiment. This log file is further updated during run-time logging events as they are happening. The Robolab program file and all other files produced by the compiler are located in the user experiment directory which helps the user to track their many experiments more easily. The system global configuration files and the code are located in a global area and thus make the maintenance of the system and its environment easier.

EXAMPLE 1 EXPERIMENTAL IMPLEMENTATION

To check the viability of the Y operation to implement basic editing operations, the editing steps depicted in Figure 2 were applied to the editing of a 700bp molecule containing the GaI lO promoter in yeast into 5 variants (Table 1). The planning and validation of the chemical constraint validation were performed the using DNA Editor software constructed according to the present invention. The output included the required primers (Table 2) and a target construction plan as described in greater detail below, which graphically specify assembly process and the resulting reactions plan. The sequence of the desired edit target (see target sequences in Table 1 ) was achieved within the first iteration of the protocol with no need for error correction. Simple de novo synthesis is expected to yield an error rate of about 1/400 (starting from purified oligos which result in additional cost) in the first iteration, which will require about 20 clones for each of the target sequences in order to achieve error- free sequence with probability of 95%. Thus the results demonstrate that editing an existing DNA molecule one can significantly improve the time of production, the cost of reagents and the effort involved in error-correction compared to de novo DNA synthesis.

Synthetic process description - Figure 6A depicts an exemplary editing protocol map in the form of a binary tree 600 as provided by a preferred embodiment of the system and method of the present invention, for performing the edit processes described in this Example. Optionally and preferably, the leaves of the binary protocol tree comprise either existing DNA input fragments NFl, preferably having valid PCR primers, or synthetic oligos DNA input sequence O9. Each internal node corresponds to a dsDNA intermediate N3, N18, N9, Nil product that can be built using a Y operation, as described above, from its two sons, which result in a simple iterative protocol. The root of the tree is the target molecule optionally comprising at least one or more targets Tl, T2, T3, T4, T5. Boxes comprising a light upward diagonal pattern represent certain parts of a given input molecule. The dotted patterned box represents an oligonucleotide which is used to insert a sequence which is not available in the input molecules and divot patterned boxes represent intermediate nodes used solely for the construction. The

arrow color (white or black) indicates whether the source molecule (located at the tail of the arrow) is composed as the left or the right side in the target molecule (located at the head).

Figure 6B is of a diagram depicting various editing operations performed on the input molecule NATIVE (see table 1). An additional input molecule, GAL80 (see table 1), is used to demonstrate substitution. In the output section, five editing operations are demonstrated. Each colored ellipse designates a part of an input molecule, identified by the color, and the numbers are coordinates inside the input molecule, in nucleotides.

Materials & Methods

Phosphorylation:

300 pmol of single stranded DNA in a 50 μl reaction containing 70 mM Tris-HCl,

10 mM MgCl ₂ , 7 mM dithiothreitol, pH 7.6 at 37 ⁰ C, 1 mM ATP & 10 units T4 Polynucleotide Kinase (NEB). Reaction is incubated at 37 ⁰ C for 30 min, then at 42 ⁰ C for 10 min and inactivated at 65 ⁰ C for 20 min. Elongation:

1 pmol of single stranded DNA of each progenitor in a 25 μl reaction containing 2.5μl of 1OX ABGene Thermo-Start Standard Buffer (no MgCl ₂ ), 1.5 mM MgCl ₂ , 200μM of each of dNTPs, 4 units Thermo-Start DNA Polymerase (ABGene). Thermal Cycler program is: Enzyme activation at 95 ⁰ C for 15 min, cooling at 0.1°C/sec to 62 ⁰ C, and holding for 10 sec, elongation at 72 ⁰ C for 10 min. PCR: 1-0.1 fmol template, 10 pmol of each primer in a 32.5 μl reaction containing 6OmM TRIS-Cl, 6mM (NH ₄ ) ₂ SO _4; 1OmM KCl, 2mM MgSO ₄ , 250μM of each of dNTPs, 1.875U of AccuSure DNA Polymerase (BioLine), SYBR Green diluted 1 :50,000. Thermal Cycler program is: 1 X Enzyme activation at 95 ⁰ C 10 min. 20 X Denaturation 95 ⁰ C for lOsec, Annealing at Tm of primers for 30sec, Extension 72 ⁰ C for lmin per kbp of template. Digestion by Lambda exonuclease:

1-5 pmol of 5' phosphorylated DNA termini in a 30μl reaction containing 67mM Glycine-KOH, 2.5mM MgCl ₂ , 0.01% Triton X-100, 5mM 1 ,4-Dithiothreitol, 5.5 units Lambda Exonuclease (Epicentre) and SYBR Green diluted 1 :50,000. Thermal

Cycler program is 37 ⁰ C for 15 min, 42 ⁰ C for 10 min, Enzyme inactivation at 65 ⁰ C for 10 min.

Mixes Preparation

Premade mixes, prepared according to the above recipes, for Phosphorylation, Elongation, PCR and Lambda Exonuclease Digestion (including enzymes and buffers) were mixed at a 5X concentration and added to the reaction.

Chemical Oligonucleotide synthesis

Oligonucleotides for all experiments were ordered by IDT or from Weizmann in- house oligonucleotides synthesis unit. All oligonucleotides undergo standard desalting.

Automated DNA Purification:

Automated DNA Purification was performed with Zymo ZR-96 DNA Purification kit in 96 well plate format using standard protocols. All centrifugations were done at 200Og for 5 min. Liquid Handling

All liquid handling except for mixes preparation were done on a Tecan Freedom

2000 robot controlled by in-house developed software. Mixes preparation were done by hand.

Capillary Electrophoresis 5' fluorescently tagged primers, tagged either with FAM or FLEX fluorophores were ordered from IDT and mixed with identical non-fluorescent primers at 1% (m/m) concentration, l μl of PCR or elongation products were mixed with 15μl pure formamide containing ladder (by ABI). Fragment analysis was done on an ABI 3130 Genetic Analyzer. Cloning

Fragments were cloned into the pGEM-T easy Vector System 1 from PROMEGA using standard procedures. Vectors containing cloned fragments were transformed into JM 109 competent cells from PROMEGAl using standard procedures. Cells were plated onto agar plates with LB+AMP antibiotics (by Hy Laboratories Ltd.) and incubated at 37°c for 1-2 days. Sequencing

Single colonies were picked manually from the plates and transferred to 50μl PBS buffer.

Plasmids were amplified in vitro using the commercial kit Templiphi (by GE Healthcare).

Amplified plasmids were sent to sequencing in house using the standard primers SP6 & T7 (see pGEM-T manual).

Table 1 : Target sequences

NATIVE

ATCGACCCGGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTT

GGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAG AGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTA ATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCC GAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGT CTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGC TCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGG AAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATTAACGAATCA AATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCT GGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATA TAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTC AGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAAT TGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGT TAATTAAGGATCCATCGA GAL80 CCTTTCTTCTCTCCCCTGCAATATAATAGTTTAATTCTAATATTAATAAT ATCCTATATTTTCTTCATTTACCGGCGCACTCTCGCCCGAACGACCTCAA AATGTCTGCTACATTCATAATAACCAAAAGCTCATAACTTTTTTTTTTGA EDTl Tl

ATCGACCCGGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTT GGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAG AGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTA ATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCC GAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGT CTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGC TCCGTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGG TAATGCCATGTAATATGATTATTAAAACAATAAAGATTCTACAATACTA GCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACA AACCTTCAAATTAACGAATCAAATTAACAACCATAGGATGATAATGCG ATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGA TTTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCACTT TAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCAAATGT CATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGT CAAGGAGAAAAAACTATAATGTTAATTAAGGATCCATCGA EDT1_T2

ATCGACCCGGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTT GGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACC

ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAG AGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTA ATACGCTTAACTGCTCATTGCTATATTGAAGTAAACAATAAAGATTCTA CAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGG CCCCACAAACCTTCAAATTAACGAATCAAATTAACAACCATAGGATGA TAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAA GCGATGATTTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATA ACCACTTTAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATT CAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACT TTAACGTCAAGGAGAAAAAACTATAATGTTAATTAAGGATCCATCGA

EDTl _T3

ATCGACCCGGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTT

GGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAG AGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTA ATACGCTTAACTGCTCATTGCTATATTGAAGTAAACAATAAAGATTCTA CAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGG CCCCACAAACCTTCAAATTAACGAATCAAATTAACAACCATAGGATGA TAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAA GCGATGATTTTTGATCTATTAACGGATTAGAAGCCGCCGAGCGGGCGAC AGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTC GCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGCAGATAT ATAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTT CAGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAA TTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATG TTAATTAAGGATCCATCGA

EDT1_T4 ATCGACCCGGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTT GGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAG AGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTA ATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCC GAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGT CTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGC TCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGG AAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATTAACGAATCA AATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCT GGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACGGATTAG AAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTG CGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCG CCGCACTGCTCCGCAGATATATAAATGGAAAAGCTGCATAACCACTTTA ACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCAAATGTCA TAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCA AGGAGAAAAAACTATAATGTTAATTAAGGATCCATCGA

EDT1JT5

ATCGACCCGGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTT GGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACC

λJBjqi| JUEUBλ |EuojEuiquio3 joj uiq)uoS]γ Buμipg VMQ

Z 31 JIMVXη

sαauiuj :z 3}qv± ςi

VODIVDDIVOOWIIVVHOIWIVIDWWWOVOOWDIODWIII

DVl VlDlDDVl Vl WllOHWWWDWDl VlOVVWl VDlOl VWDl

IVllDllOVllVlOlllOVDllllVDWDlllDVlWlOWlllDVOOW

IVDOIDOWWOOI WVlVl Vl VOVDWIl VIDIVOIHIIVOI VODOV Ol

VODOVDl VVJLL WlOOOOlDlllVHDDOVHHllOVllVODOl WlV

OIVOOVIVDDWDWH VVVDl WODWIl WVDHDDWVDVDDDDO

OlDDWlOVDOOllVWWOOVOWOl VHOOl V1111D0V1DV1 VVDV

1011 VOVVVl VVVVDDWl WlV

DHVDVIDOIDIOIVWVDIDDVODWODDDODIDIDVDODOODDVIJLL

VD11D1111 Vl VlDDl Vl VVl VVIl Vl VVlDIl VVHIOVI Wl VlVVDO

IDDDDIDIDIIDIHDDVIOWOHVIVIDOIIVDIDOIDWIIDODVIV

VlOVDlllDWOOlllDlDllDDWVVVWlDDOVllDlVllVDDDDOV

OWVIOIVWOOIOIIOI Vl VHDVllDl VVlDl VlVDDl VIVDVlVlV

Z9ϊ00/800ση/13d 9ϊ08/.0/600Z OλV

This Example relates to the application of the above-described method for a combinatorial variant library, by using the divide and conquer approach that extends the above described DNA editing method. Since a typical variant library contains many DNA fragments that are shared between the library variants, it is desired to produce shared fragments only once and re-use them as larger building blocks for building other variants. A divide and conquer algorithm computes an efficient protocol that minimizes the cost, time and effort of library production by maximizing the reuse of shared library components. The result of the algorithm is a compact protocol that converges towards the largest shared fragments of the entire variants library. Those large shared fragments are built only once and then reused as a building block for the construction of the final targets.

Given a set of target DNA sequences which are combinatorial variant library of one original template, the method provides an efficient D&C edit protocol for the construction of all the targets while maximizing reuse of shared library components. Algorithm:

Input: a set of target sequences T which are variant library of one original sequence and existing sequences S.

Pre-processing:

Go over all the sub-sequences of all the variants that are larger than or equal to a given length MIN_FRAGMENT_SIZE. For each sub-sequence count the number of occurrences in the final products.

For each sequence compute a score which reflects the amount of final library products. The size multiplied by the number of occurrences.

FRAGMENT SCORE = FRAGMENT SIZE * FRAGMENT NUMBER. Store the fragment score in a FRAGMENTS DB

Divide & Conquer:

For each of the sequences in T:

• The target sequence is given as the initial input for a divide and conquers recursive protocol planning subroutine.

• At each recursive step the target sequence is divided at each step into two adjacent parts.

• The division point is selected as follows:

o All possible division points are considered. The current candidate division is denoted by CURR CANDIDATE o Find the maximal scoring fragment in FRAGMENT DB that matches the sequence that ends in CURR CANDIDATE. This fragment is called MAX LEFT. o Find the maximal scoring fragment in FRAGMENT DB that matches the sequence that starts after the CURR CANDIDATE.

This fragment is called MAX RIGHT. o The score of the current point CURR CANDIDATE is calculated by the sum of the two adjacent maximal fragments.

CANDIDATE SCORE = MAX LEFT + MAX RIGHT o The CANDIDATE SCORE is registered in a list SCORE LIST for each candidate point. o The SCORE LIST is sorted to determine the priority of each point for division.

• Thus each candidate point is scored according to the sum of the two maximal fragments scores that starts and ends exactly at this point. Thus points which are exactly adjacent to fragments which have large score (reflecting the fact that they are shared by many of the final products) will be preferred for division in many of the target that share them (See intuitive explanation below).

• The selected division point is validated for fulfilling the chemical constraints of the basic edit step (See basic chemical step in the DNA edit paper). • The recursive division ends when one of the following occurs: o The current fragment was already planned (as part of previous target for example) o The current fragment is found within an existing fragment from

S. o The current fragment is in the size of an oligo that can be synthesized.

• If a valid division could not be found (for example, due to chemical constraints) the recursion subroutine returns failure and the next best scoring candidate is selected.

• Each division result in two nodes in a graph that are connected to the node which was divided (their parent node). The division continues recursively for the two new nodes.

• In case of success the graph node ID of the current sub-target is returned to the calling level.

The output of the algorithm is a set of instructions, preferably in the form of a directed graph which describes the protocol to build the library. Each node in the graph corresponds to DNA fragment. Each node has at most two incoming edges that describe from which nodes it is assembled. A shared component has more than one outgoing edge, reflecting the fact that it is reused.

EXAMPLE 3

A protein-variant library for protein design This Example relates to creating a library of different variations on a particular protein, for example for protein design. For example the DNA of a gene coding for a protein with a known function and structure may be available; however, it may be desired to improve the protein's activity (for example, its catalytic activity or binding affinity). It is possible to screen for a variant of the original protein by replacing the amino acids near the known sites of activity. Typically, desired amino acid mutations occur along the entire length of the DNA coding for the protein, which makes changing several amino acids difficult.

Moreover, predicting the result of mutations is difficult, and often it is required to make several mutations before finding a suitable protein.

This example demonstrates the ability of the method of the present invention, in this embodiment, to make up to 12 mutations concurrently and thus efficiently and quickly with only 5 iterations of the construction process (see Figure 7B). In this Example, one such gene, DRI (see table 3, DRIl NFl ), which is 813bp long, was used to create a library of 1 1 mutants which were later used for the optimization of its affinity. Figure 7a illustrates the desired sequences as a

function of the input gene, each with 2 to 12 amino acid substitutions. The editing protocol map (Fig 7b) has a depth of 5 levels and demonstrates both efficient utilization of the available DNA molecule as well as the sharing of library components among different variants. Since the Y operation can use as input a fragment embedded in a longer sequence, a further optimization is possible, by eliminating graph nodes that represent a fragments contained in other nodes, and using the containing node as the source of the Y operation instead (Fig. 7c). Figure 7D demonstrates the difficulty of manually performing these editing tasks by showing the full reaction protocol, including all the primers that take part in the process. This complex graph is translated into a robot control program that when executed performs the specified edit protocol. In reality, each arrow in the graph is translated into one or more reagent transfer in the robot program.

The complete set of sequences is shown in Table 3 below. The sequences were created according to the methods described in Figure 7D and the methods described with regard to Example 1 ; upon sequencing, no errors were found (data not shown).

Table 3 - DRI example sequences

DRIl NFl is the input molecule (DRI). DRIl Tl through DRIl Tl 1 correspond to Vl through VI l . DRI I Pl through DRI1 P69 are the oligos used by the protocol.

Z9ϊ00/800πi/13d 9ϊ08L0/600Z OλV

Example 4 A protein-chimera library

In another example, two protein enzymes share extensive sequence homology, yet differ in their substrate specificity. In order to determine which part of the enzymes determines the specificity protein, chimeras may optionally be created, combining different parts from the two proteins. As in any type of search, the speed at which the right position is found is dependent on the amount of

variants constructed and tested, given that an efficient assay for measure substrate specificity is available.

For this Example, two genes, CER5 and CER2, were used, recombining them in six different ways and making two mutants in one of them. For details see Figures 8A and 8B. Briefly, Figure 8A shows desired recombinations of the two input genes, including two mutants on one of the input genes. In addition, restriction sites were added as flanking regions to each target to facilitate downstream application of the constructs. Figure 8B shows the construction plan. Note that adding flanking regions as well as making small mutations within sequence do not require additional Y operation and can be incorporated into the primers.

In this example, no error correction step was required. Out of 8 constructs, only one contained an error and a second clone tested was error free.

This example demonstrates the ability to reuse existing DNA to avoid costly error correction as the result is often error free. It thus demonstrates the power of the system to achieve editing of DNA completely in vitro and to combine large pieces of existing DNA.

Materials and Methods

The complete set of sequences is shown in Table 4 below. The sequences were created according to the methods depicted in Figure 8B and the methods described with regard to Example 1 ; upon sequencing, no errors were found (data not shown).

Table 4 — CER example sequences

CER NFl / NF2 are the input molecules (CER5 / CER2 respectively). CER Tl through CER T8 are the eight target molecules (referred to as C1-C8 in the figures). CER Pl through CER P25 are the oligos used by the protocol.

Size Sequence

CER_P1 29 CCGAATTCGCGACAGCAGCGCAGGGACCC

CER_P2 35 TGGTGGCCACGTGATGCACAAACATGATCAGGAAG

CER_P3 32 TGTTTGTGCATCACGTGGCCACCATCATTCTC

CER_P4 30 CCCTCGAGTCAGTCATTCTTACGATGGTTG

CER_P5 35 GTTCCTTGAAGTCCTTTCTTTTAATGTCTGTAAAC

CER P6 48 CAGTTTACAGACATTAAAAGAAAGGACTTCAAGGAACA

GATCATCCAC CER_P7 34 AAGTTCAATCATATAATAGTGATAAAGCCCACTT

TTCAAGTGGGCTTTATCACTATTATATGATTGAACTTTC U - CTTCTACTGGTCC

CER_P9 31 GCAATCAGATAAAATGTGAATCTCCACATGC

., _D GCATGTGGAGATTCACATTTTATCTGATTGCCTTCATT

CER_P10 46 _GCCGGCAT oc _{D D} i _{λ oo} CCCTCGAGTTACTCTTCAGCCCAGTAGCTGCCTCCCA tbK_r ι I Jo -p

CER_P12 37 ATATACATAAGTAAAATGTGAATCTCCAGCTGGCTTC AGCCAGCTGGAGATTCACATTTTACTTATGTATATTCT

^Ut - ^{1 J 0} ^ GCTATGGAATTAGA

CER_P14 30 CCGAATTCCTCCAGACCTTGTATGATTACT

CER_P15 24 CGGTCCTGGTTCCTCCGATGGCGA

CER_P16 32 CGCCATCGGAGGAACCAGGACCGGCCCAGTCT

CER_P17 22 CCGAATTCCTCCAGACCTTGTA

_{OCD D} - _{I Q co} . GATACTTCTTTGAGCGATACGTGGCTACACCACTGGCT LbK_P18 53 _{GCCCTCTTGAACATA}

CER_P19 33 CCCTCGAGTCAGTCATTCTTACGATGGTTGTTA

CER_P20 32 AGCCACGTATCGCTCAAAGAAGTATCGAACGA

CER_P21 26 CCACAATGACTCTCATGCCGGCAATG GCCGGCATGAGAGTCATTGTGGATAAACCCTGGTT , oc _{D D} oo _{λ I} GATACTTCTTTGAGCGATACGTGGCTACACCACTGGCT CER_P23 43 _GCCCT

CER_P24 19 TCTCATGCCGGCAATGAAG

CER_P25 33 TAGCCACGTATCGCTCAAAGAAGTATCGAACGA

GCGACAGCAGCGCAGGGACCCCTAAGCTTGCTGTGG

GGCTGGCTGTGGAGCGAGCGCTTCTGGCTACCCGAG

AACGTGAGCTGGGCTGATCTGGAGGGGCCGGCCGAC

GGCTACGGTTACCCCCGCGGCCGGCACATCCTCTCG

GTGTTCCCGCTGGCGGCGGGCATCTTCTTCGTGAGGC

TGCTCTTCGAGCGATTTATTGCCAAACCCTGTGCACTC

TGTATTGGCATCGAGGACAGTGGTCCTTATCAGGCCC

AACCCAATGCCATCCTTGAAAAGGTGTTCATATCTATT

ACCAAGTATCCTGATAAGAAAAGGCTGGAGGGCCTGT

CAAAGCAGCTGGATTGGAATGTCCGAAAAATCCAATG

CTGGTTTCGCCATCGGAGGAATCAGGACAAGCCCCCA

CER_NF1 1176 ACGCTTACTAAATTCTGTGAAAGCATGTGGAGATTCAC ATTTTATTTATGTATATTCTGCTATGGAATTAGATTTCTC TGGTCGTCACCTTGGTTCTGGGACATCCGACAGTGCT GGCATAACTATCCATTTCAGCCTCTTTCAAGTGGGCTT TATCACTATTATATCATGGAATTGGCCTTCTATTGGTCC CTTATGTTTTCTCAGTTTACAGACATTAAAAGAAAGGAC TTCCTGATCATGTTTGTGCATCACTTGGTCACCATTGG GCTTATCTCCTTCTCCTACATCAACAATATGGTTCGAG TGGGAACTCTGATCATGTGTCTACATGATGTCTCAGAC TTCTTGCTGGAGGCAGCCAAACTGGCCAATTATGCCA AGTATCAGCGGCTCTGTGACACCCTTTTTGTGATCTTC AGTGCTGTTTTTATGGTTACACGACTAGGAATCTATCC

ATTCTGGATTCTGAACACGACCCTCTTTGAGAGTTGGG AGATAATCGGGCCTTATGCTTCATGGTGGCTCCTCAAT GGCCTGCTGCTGACCCTACAGCTTCTGCATGTCATCT GGTCCTACCTAATTGCACGGATTGCTTTGAAAGCCTTG ATCAGGGGAAAGGTATCGAAGGATGATCGCAGTGATG TGGAGAGCAGCTCAGAGGAAGAAGATGTGACCACCTG CACAAAAAGTCCCTGTGACAGTAGCTCCAGCAATGGT GCCAATCGGGTGAATGGTCACATGGGAGGCAGCTACT GGGCTGAAGAGTAA

CTCCAGACCTTGTATGATTACTTCTGGTGGGAACGTCT GTGGCTGCCTGTGAACTTGACCTGGGCCGATCTAGAA GACCGAGATGGACGTGTCTACGCCAAAGCCTCAGATC TCTATATCACGCTGCCCCTGGCCTTGCTCTTCCTCATC GTTCGATACTTCTTTGAGCTGTACGTGGCTACACCACT GGCTGCCCTCTTGAACATAAAGGAGAAAACTCGGCTG CGGGCACCTCCCAACGCCACCTTGGAACATTTCTACC TGACCAGTGGCAAGCAGCCCAAGCAGGTGGAAGTAG AGCTTTTGTCCCGGCAGAGCGGGCTCTCTGGCCGCCA GGTAGAGCGTTGGTTCCGTCGCCGCCGCAACCAGGA CCGGCCCAGTCTCCTCAAGAAGTTCCGAGAAGCCAGC TGGAGATTCACATTTTACCTGATTGCCTTCATTGCCGG CATGGCCGTCATTGTGGATAAACCCTGGTTCTATGACA TGAAGAAAGTTTGGGAGGGATATCCCATACAGAGCAC TATCCCTTCCCAGTATTGGTACTACATGATTGAACTTTC CER_NF2 1140 CTTCTACTGGTCCCTGCTCTTCAGCATTGCCTCTGATG TCAAGCGAAAGGATTTCAAGGAACAGATCATCCACCAT GTGGCCACCATCATTCTCATCAGCTTTTCCTGGTTTGC CAATTACATCCGAGCTGGGACTCTAATCATGGCTCTGC ATGACTCTTCCGATTACCTGCTGGAGTCAGCCAAGAT GTTTAACTACGCGGGATGGAAGAACACCTGCAACAAC ATCTTCATCGTCTTCGCCATTGTTTTTATCATCACCCGA CTGGTCATCCTGCCCTTCTGGATCCTGCATTGCACCCT GGTGTACCCACTGGAGCTCTATCCTGCCTTCTTTGGCT ATTACTTCTTCAATTCCATGATGGGAGTTCTACAGCTG CTGCATATCTTCTGGGCCTACCTCATTTTGCGCATGGC CCACAAGTTCATAACTGGAAAGCTGGTAGAAGATGAA CGCAGTGACCGGGAAGAAACAGAGAGCTCAGAGGGG GAGGAGGCTGCAGCTGGGGGAGGAGCAAAGAGCCG GCCCCTAGCCAATGGCCACCCCATCCTCAATAACAAC CATCGTAAGAATGACTGA

CCGAATTCGCGACAGCAGCGCAGGGACCCCTAAGCTT GCTGTGGGGCTGGCTGTGGAGCGAGCGCTTCTGGCT ACCCGAGAACGTGAGCTGGGCTGATCTGGAGGGGCC GGCCGACGGCTACGGTTACCCCCGCGGCCGGCACAT CCTCTCGGTGTTCCCGCTGGCGGCGGGCATCTTCTTC - GTGAGGCTGCTCTTCGAGCGATTTATTGCCAAACCCT

GTGCACTCTGTATTGGCATCGAGGACAGTGGTCCTTA TCAGGCCCAACCCAATGCCATCCTTGAAAAGGTGTTC ATATCTATTACCAAGTATCCTGATAAGAAAAGGCTGGA GGGCCTGTCAAAGCAGCTGGATTGGAATGTCCGAAAA

ATCCAATGCTGGTTTCGCCATCGGAGGAATCAGGACA AGCCCCCAACGCTTACTAAATTCTGTGAAAGCATGTGG AGATTCACATTTTATTTATGTATATTCTGCTATGGAATT AGATTTCTCTGGTCGTCACCTTGGTTCTGGGACATCCG ACAGTGCTGGCATAACTATCCATTTCAGCCTCTTTCAA GTGGGCTTTATCACTATTATATCATGGAATTGGCCTTC TATTGGTCCCTTATGTTTTCTCAGTTTACAGACATTAAA AGAAAGGACTTCCTGATCATGTTTGTGCATCACGTGGC CACCATCATTCTCATCAGCTTTTCCTGGTTTGCCAATTA CATCCGAGCTGGGACTCTAATCATGGCTCTGCATGAC TCTTCCGATTACCTGCTGGAGTCAGCCAAGATGTTTAA CTACGCGGGATGGAAGAACACCTGCAACAACATCTTC ATCGTCTTCGCCATTGTTTTTATCATCACCCGACTGGT CATCCTGCCCTTCTGGATCCTGCATTGCACCCTGGTG TACCCACTGGAGCTCTATCCTGCCTTCTTTGGCTATTA CTTCTTCAATTCCATGATGGGAGTTCTACAGCTGCTGC ATATCTTCTGGGCCTACCTCATTTTGCGCATGGCCCAC AAGTTCATAACTGGAAAGCTGGTAGAAGATGAACGCA GTGACCGGGAAGAAACAGAGAGCTCAGAGGGGGAGG AGGCTGCAGCTGGGGGAGGAGCAAAGAGCCGGCCCC TAGCCAATGGCCACCCCATCCTCAATAACAACCATCGT AAGAATGACTGACTCGAGGG

CCGAATTCGCGACAGCAGCGCAGGGACCCCTAAGCTT GCTGTGGGGCTGGCTGTGGAGCGAGCGCTTCTGGCT ACCCGAGAACGTGAGCTGGGCTGATCTGGAGGGGCC GGCCGACGGCTACGGTTACCCCCGCGGCCGGCACAT CCTCTCGGTGTTCCCGCTGGCGGCGGGCATCTTCTTC GTGAGGCTGCTCTTCGAGCGATTTATTGCCAAACCCT GTGCACTCTGTATTGGCATCGAGGACAGTGGTCCTTA TCAGGCCCAACCCAATGCCATCCTTGAAAAGGTGTTC ATATCTATTACCAAGTATCCTGATAAGAAAAGGCTGGA GGGCCTGTCAAAGCAGCTGGATTGGAATGTCCGAAAA ATCCAATGCTGGTTTCGCCATCGGAGGAATCAGGACA AGCCCCCAACGCTTACTAAATTCTGTGAAAGCATGTGG AGATTCACATTTTATTTATGTATATTCTGCTATGGAATT AGATTTCTCTGGTCGTCACCTTGGTTCTGGGACATCCG ^υ - ACAGTGCTGGCATAACTATCCATTTCAGCCTCTTTCAA

GTGGGCTTTATCACTATTATATCATGGAATTGGCCTTC ' TATTGGTCCCTTATGTTTTCTCAGTTTACAGACATTAAA AGAAAGGACTTCAAGGAACAGATCATCCACCATGTGG CCACCATCATTCTCATCAGCTTTTCCTGGTTTGCCAAT TACATCCGAGCTGGGACTCTAATCATGGCTCTGCATG ACTCTTCCGATTACCTGCTGGAGTCAGCCAAGATGTTT AACTACGCGGGATGGAAGAACACCTGCAACAACATCT TCATCGTCTTCGCCATTGTTTTTATCATCACCCGACTG GTCATCCTGCCCTTCTGGATCCTGCATTGCACCCTGG TGTACCCACTGGAGCTCTATCCTGCCTTCTTTGGCTAT TACTTCTTCAATTCCATGATGGGAGTTCTACAGCTGCT GCATATCTTCTGGGCCTACCTCATTTTGCGCATGGCCC ACAAGTTCATAACTGGAAAGCTGGTAGAAGATGAACG

CAGTGACCGGGAAGAAACAGAGAGCTCAGAGGGGGA

GGAGGCTGCAGCTGGGGGAGGAGCAAAGAGCCGGC

CCCTAGCCAATGGCCACCCCATCCTCAATAACAACCAT

CGTAAGAATGACTGACTCGAGGG

CCGAATTCGCGACAGCAGCGCAGGGACCCCTAAGCTT

GCTGTGGGGCTGGCTGTGGAGCGAGCGCTTCTGGCT

ACCCGAGAACGTGAGCTGGGCTGATCTGGAGGGGCC

GGCCGACGGCTACGGTTACCCCCGCGGCCGGCACAT

CCTCTCGGTGTTCCCGCTGGCGGCGGGCATCTTCTTC

GTGAGGCTGCTCTTCGAGCGATTTATTGCCAAACCCT

GTGCACTCTGTATTGGCATCGAGGACAGTGGTCCTTA

TCAGGCCCAACCCAATGCCATCCTTGAAAAGGTGTTC

ATATCTATTACCAAGTATCCTGATAAGAAAAGGCTGGA

GGGCCTGTCAAAGCAGCTGGATTGGAATGTCCGAAAA

ATCCAATGCTGGTTTCGCCATCGGAGGAATCAGGACA

AGCCCCCAACGCTTACTAAATTCTGTGAAAGCATGTGG

AGATTCACATTTTATTTATGTATATTCTGCTATGGAATT

AGATTTCTCTGGTCGTCACCTTGGTTCTGGGACATCCG

ACAGTGCTGGCATAACTATCCATTTCAGCCTCTTTCAA

GTGGGCTTTATCACTATTATATGATTGAACTTTCCTTCT

° - ACTGGTCCCTGCTCTTCAGCATTGCCTCTGATGTCAAG

CGAAAGGATTTCAAGGAACAGATCATCCACCATGTGG CCACCATCATTCTCATCAGCTTTTCCTGGTTTGCCAAT TACATCCGAGCTGGGACTCTAATCATGGCTCTGCATG ACTCTTCCGATTACCTGCTGGAGTCAGCCAAGATGTTT AACTACGCGGGATGGAAGAACACCTGCAACAACATCT TCATCGTCTTCGCCATTGTTTTTATCATCACCCGACTG GTCATCCTGCCCTTCTGGATCCTGCATTGCACCCTGG TGTACCCACTGGAGCTCTATCCTGCCTTCTTTGGCTAT TACTTCTTCAATTCCATGATGGGAGTTCTACAGCTGCT GCATATCTTCTGGGCCTACCTCATTTTGCGCATGGCCC ACAAGTTCATAACTGGAAAGCTGGTAGAAGATGAACG CAGTGACCGGGAAGAAACAGAGAGCTCAGAGGGGGA GGAGGCTGCAGCTGGGGGAGGAGCAAAGAGCCGGC CCCTAGCCAATGGCCACCCCATCCTCAATAACAACCAT CGTAAGAATGACTGACTCGAGGG CCGAATTCGCGACAGCAGCGCAGGGACCCCTAAGCTT GCTGTGGGGCTGGCTGTGGAGCGAGCGCTTCTGGCT ACCCGAGAACGTGAGCTGGGCTGATCTGGAGGGGCC GGCCGACGGCTACGGTTACCCCCGCGGCCGGCACAT CCTCTCGGTGTTCCCGCTGGCGGCGGGCATCTTCTTC GTGAGGCTGCTCTTCGAGCGATTTATTGCCAAACCCT GTGCACTCTGTATTGGCATCGAGGACAGTGGTCCTTA otκ_ ι 4 π ou jcAGGcccAAccCAATGCCATCCTTGAAAAGGTGTTC ATATCTATTACCAAGTATCCTGATAAGAAAAGGCTGGA GGGCCTGTCAAAGCAGCTGGATTGGAATGTCCGAAAA ATCCAATGCTGGTTTCGCCATCGGAGGAATCAGGACA AGCCCCCAACGCTTACTAAATTCTGTGAAAGCATGTGG AGATTCACATTTTATCTGATTGCCTTCATTGCCGGCAT GGCCGTCATTGTGGATAAACCCTGGTTCTATGACATGA

AGAAAGTTTGGGAGGGATATCCCATACAGAGCACTAT

CCCTTCCCAGTATTGGTACTACATGATTGAACTTTCCT

TCTACTGGTCCCTGCTCTTCAGCATTGCCTCTGATGTC

AAGCGAAAGGATTTCAAGGAACAGATCATCCACCATGT

GGCCACCATCATTCTCATCAGCTTTTCCTGGTTTGCCA

ATTACATCCGAGCTGGGACTCTAATCATGGCTCTGCAT

GACTCTTCCGATTACCTGCTGGAGTCAGCCAAGATGTT

TAACTACGCGGGATGGAAGAACACCTGCAACAACATC

TTCATCGTCTTCGCCATTGTTTTTATCATCACCCGACT

GGTCATCCTGCCCTTCTGGATCCTGCATTGCACCCTG

GTGTACCCACTGGAGCTCTATCCTGCCTTCTTTGGCTA

TTACTTCTTCAATTCCATGATGGGAGTTCTACAGCTGC

TGCATATCTTCTGGGCCTACCTCATTTTGCGCATGGCC

CACAAGTTCATAACTGGAAAGCTGGTAGAAGATGAAC

GCAGTGACCGGGAAGAAACAGAGAGCTCAGAGGGGG

AGGAGGCTGCAGCTGGGGGAGGAGCAAAGAGCCGGC

CCCTAGCCAATGGCCACCCCATCCTCAATAACAACCAT

CGTAAGAATGACTGACTCGAGGG

CCGAATTCCTCCAGACCTTGTATGATTACTTCTGGTGG

GAACGTCTGTGGCTGCCTGTGAACTTGACCTGGGCCG

ATCTAGAAGACCGAGATGGACGTGTCTACGCCAAAGC

CTCAGATCTCTATATCACGCTGCCCCTGGCCTTGCTCT

TCCTCATCGTTCGATACTTCTTTGAGCTGTACGTGGCT

ACACCACTGGCTGCCCTCTTGAACATAAAGGAGAAAA

CTCGGCTGCGGGCACCTCCCAACGCCACCTTGGAACA

TTTCTACCTGACCAGTGGCAAGCAGCCCAAGCAGGTG

GAAGTAGAGCTTTTGTCCCGGCAGAGCGGGCTCTCTG

GCCGCCAGGTAGAGCGTTGGTTCCGTCGCCGCCGCA

ACCAGGACCGGCCCAGTCTCCTCAAGAAGTTCCGAGA

AGCCAGCTGGAGATTCACATTTTACTTATGTATATTCT

GCTATGGAATTAGATTTCTCTGGTCGTCACCTTGGTTC

TGGGACATCCGACAGTGCTGGCATAACTATCCATTTCA

GCCTCTTTCAAGTGGGCTTTATCACTATTATATCATGG

AATTGGCCTTCTATTGGTCCCTTATGTTTTCTCAGTTTA u tκ_ i o H ^b B _{CAGACATTAAAAGAAAGGACTTCCTGATCATGTTTGTG}

CATCACTTGGTCACCATTGGGCTTATCTCCTTCTCCTA

CATCAACAATATGGTTCGAGTGGGAACTCTGATCATGT

GTCTACATGATGTCTCAGACTTCTTGCTGGAGGCAGC

CAAACTGGCCAATTATGCCAAGTATCAGCGGCTCTGT

GACACCCTTTTTGTGATCTTCAGTGCTGTTTTTATGGTT

ACACGACTAGGAATCTATCCATTCTGGATTCTGAACAC

GACCCTCTTTGAGAGTTGGGAGATAATCGGGCCTTAT

GCTTCATGGTGGCTCCTCAATGGCCTGCTGCTGACCC

TACAGCTTCTGCATGTCATCTGGTCCTACCTAATTGCA

CGGATTGCTTTGAAAGCCTTGATCAGGGGAAAGGTAT

CGAAGGATGATCGCAGTGATGTGGAGAGCAGCTCAGA

GGAAGAAGATGTGACCACCTGCACAAAAAGTCCCTGT

GACAGTAGCTCCAGCAATGGTGCCAATCGGGTGAATG

GTCACATGGGAGGCAGCTACTGGGCTGAAGAGTAACT

CGAGGG

CCGAATTCGCGACAGCAGCGCAGGGACCCCTAAGCTT

GCTGTGGGGCTGGCTGTGGAGCGAGCGCTTCTGGCT

ACCCGAGAACGTGAGCTGGGCTGATCTGGAGGGGCC

GGCCGACGGCTACGGTTACCCCCGCGGCCGGCACAT

CCTCTCGGTGTTCCCGCTGGCGGCGGGCATCTTCTTC

GTGAGGCTGCTCTTCGAGCGATTTATTGCCAAACCCT

GTGCACTCTGTATTGGCATCGAGGACAGTGGTCCTTA

TCAGGCCCAACCCAATGCCATCCTTGAAAAGGTGTTC

ATATCTATTACCAAGTATCCTGATAAGAAAAGGCTGGA

GGGCCTGTCAAAGCAGCTGGATTGGAATGTCCGAAAA

ATCCAATGCTGGTTTCGCCATCGGAGGAACCAGGACC

GGCCCAGTCTCCTCAAGAAGTTCCGAGAAGCCAGCTG

GAGATTCACATTTTACCTGATTGCCTTCATTGCCGGCA

TGGCCGTCATTGTGGATAAACCCTGGTTCTATGACATG

AAGAAAGTTTGGGAGGGATATCCCATACAGAGCACTA

TCCCTTCCCAGTATTGGTACTACATGATTGAACTTTCC o t κ_ o ou _{TTCTACTGGTCCCTGCTCTTCAGCATTGCCTCTGATGT}

CAAGCGAAAGGATTTCAAGGAACAGATCATCCACCAT GTGGCCACCATCATTCTCATCAGCTTTTCCTGGTTTGC CAATTACATCCGAGCTGGGACTCTAATCATGGCTCTGC ATGACTCTTCCGATTACCTGCTGGAGTCAGCCAAGAT GTTTAACTACGCGGGATGGAAGAACACCTGCAACAAC ATCTTCATCGTCTTCGCCATTGTTTTTATCATCACCCGA CTGGTCATCCTGCCCTTCTGGATCCTGCATTGCACCCT GGTGTACCCACTGGAGCTCTATCCTGCCTTCTTTGGCT ATTACTTCTTCAATTCCATGATGGGAGTTCTACAGCTG CTGCATATCTTCTGGGCCTACCTCATTTTGCGCATGGC CCACAAGTTCATAACTGGAAAGCTGGTAGAAGATGAA CGCAGTGACCGGGAAGAAACAGAGAGCTCAGAGGGG GAGGAGGCTGCAGCTGGGGGAGGAGCAAAGAGCCG GCCCCTAGCCAATGGCCACCCCATCCTCAATAACAAC CATCGTAAGAATGACTGACTCGAGGG CCGAATTCCTCCAGACCTTGTATGATTACTTCTGGTGG GAACGTCTGTGGCTGCCTGTGAACTTGACCTGGGCCG ATCTAGAAGACCGAGATGGACGTGTCTACGCCAAAGC CTCAGATCTCTATATCACGCTGCCCCTGGCCTTGCTCT TCCTCATCGTTCGATACTTCTTTGAGCGATACGTGGCT ACACCACTGGCTGCCCTCTTGAACATAAAGGAGAAAA CTCGGCTGCGGGCACCTCCCAACGCCACCTTGGAACA TTTCTACCTGACCAGTGGCAAGCAGCCCAAGCAGGTG GAAGTAGAGCTTTTGTCCCGGCAGAGCGGGCTCTCTG UhK_ \ l nab GCCGCCAGGTAGAGCGTTGGTTCCGTCGCCGCCGCA ACCAGGACCGGCCCAGTCTCCTCAAGAAGTTCCGAGA AGCCAGCTGGAGATTCACATTTTACCTGATTGCCTTCA TTGCCGGCATGGCCGTCATTGTGGATAAACCCTGGTT CTATGACATGAAGAAAGTTTGGGAGGGATATCCCATAC AGAGCACTATCCCTTCCCAGTATTGGTACTACATGATT GAACTTTCCTTCTACTGGTCCCTGCTCTTCAGCATTGC CTCTGATGTCAAGCGAAAGGATTTCAAGGAACAGATCA TCCACCATGTGGCCACCATCATTCTCATCAGCTTTTCC

TGGTTTGCCAATTACATCCGAGCTGGGACTCTAATCAT GGCTCTGCATGACTCTTCCGATTACCTGCTGGAGTCA GCCAAGATGTTTAACTACGCGGGATGGAAGAACACCT GCAACAACATCTTCATCGTCTTCGCCATTGTTTTTATCA TCACCCGACTGGTCATCCTGCCCTTCTGGATCCTGCA TTGCACCCTGGTGTACCCACTGGAGCTCTATCCTGCC TTCTTTGGCTATTACTTCTTCAATTCCATGATGGGAGTT CTACAGCTGCTGCATATCTTCTGGGCCTACCTCATTTT GCGCATGGCCCACAAGTTCATAACTGGAAAGCTGGTA GAAGATGAACGCAGTGACCGGGAAGAAACAGAGAGCT CAGAGGGGGAGGAGGCTGCAGCTGGGGGAGGAGCA AAGAGCCGGCCCCTAGCCAATGGCCACCCCATCCTCA ATAACAACCATCGTAAGAATGACTGACTCGAGGG CCGAATTCCTCCAGACCTTGTATGATTACTTCTGGTGG GAACGTCTGTGGCTGCCTGTGAACTTGACCTGGGCCG ATCTAGAAGACCGAGATGGACGTGTCTACGCCAAAGC CTCAGATCTCTATATCACGCTGCCCCTGGCCTTGCTCT TCCTCATCGTTCGATACTTCTTTGAGCGATACGTGGCT ACACCACTGGCTGCCCTCTTGAACATAAAGGAGAAAA CTCGGCTGCGGGCACCTCCCAACGCCACCTTGGAACA TTTCTACCTGACCAGTGGCAAGCAGCCCAAGCAGGTG GAAGTAGAGCTTTTGTCCCGGCAGAGCGGGCTCTCTG GCCGCCAGGTAGAGCGTTGGTTCCGTCGCCGCCGCA ACCAGGACCGGCCCAGTCTCCTCAAGAAGTTCCGAGA AGCCAGCTGGAGATTCACATTTTACCTGATTGCCTTCA TTGCCGGCATGAGAGTCATTGTGGATAAACCCTGGTT CTATGACATGAAGAAAGTTTGGGAGGGATATCCCATAC AGAGCACTATCCCTTCCCAGTATTGGTACTACATGATT CER_T8 1156 GAACTTTCCTTCTACTGGTCCCTGCTCTTCAGCATTGC CTCTGATGTCAAGCGAAAGGATTTCAAGGAACAGATCA TCCACCATGTGGCCACCATCATTCTCATCAGCTTTTCC TGGTTTGCCAATTACATCCGAGCTGGGACTCTAATCAT GGCTCTGCATGACTCTTCCGATTACCTGCTGGAGTCA GCCAAGATGTTTAACTACGCGGGATGGAAGAACACCT GCAACAACATCTTCATCGTCTTCGCCATTGTTTTTATCA TCACCCGACTGGTCATCCTGCCCTTCTGGATCCTGCA TTGCACCCTGGTGTACCCACTGGAGCTCTATCCTGCC TTCTTTGGCTATTACTTCTTCAATTCCATGATGGGAGTT CTACAGCTGCTGCATATCTTCTGGGCCTACCTCATTTT GCGCATGGCCCACAAGTTCATAACTGGAAAGCTGGTA GAAGATGAACGCAGTGACCGGGAAGAAACAGAGAGCT CAGAGGGGGAGGAGGCTGCAGCTGGGGGAGGAGCA AAGAGCCGGCCCCTAGCCAATGGCCACCCCATCCTCA ATAACAACCATCGTAAGAATGACTGACTCGAGGG

REFERENCES

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4 Cirino, P., Mayer, K. & Umeno, D. Generating mutant libraries using error-prone PCR. Methods MoI Biol 231, 3-9 (2003).

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I 1 Smith, H., Hutchison, C. r., Pfannkoch, C. & Venter, J. Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci U S A 100, 15440-15445 (2003). 12 Xiong, A. et al. PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1, 791-797 (2006). 13 Horton, R., Hunt, H., Ho, S., Pullen, J. & Pease, L. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61-68 (1989).

14 Coco, W. et al. DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol 19, 354- 359 (2001).

15 Gaytan, P., Yafϊez, J., Sanchez, F., Mackie, H. & Soberόn, X. Combination of DMT-mononucleotide and Fmoc-trinucleotide phosphoramidites in oligonucleotide synthesis affords an automatable codon-level mutagenesis method. Chem Biol 5, 519- 527 (1998).

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18. Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G., Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432(7020): 1050-4 (2004).

While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made.