Editing Error Correction in Gene Synthesis (section)

== Error-removal using MutS mismatch-binding protein ==

The MutS protein is part of the bacterial MutHLS mismatch repair mechinary [38]. It detects and binds to a variety of mispaired bases and small single-strand loops in vivo. Methods have been developed that use MutS as an error-removal agent for gene synthesis. In one strategy, MutS protein from the thermophilic bacteria Thermus aquaticus was used to directly filter out full-length heteroduplexes [8]. After denaturation and reannealing, mismatch containing heteroduplexes are recognized and bound by the Taq MutS protein, which can be separated from the unbound homoduplexes (mostly error-free) by a gel-mobility shift assay (Fig. 2a). This method was reported to reduce error rate to 1 error per 3.8kb with one round of filtration, which represented several folds of improvement from raw synthesis products. Repeating the procedure one more time reportedly reduced the error rate to 1 error per 10 kb, which is good enough to ensure finding a correct sequence with a single round of cloning and sequencing. A major limitation with removing full-length heteroduplexes is that it requires a substantial fraction of the sequences to be error-free in order to survive the MutS binding filtration. Figure 2 Figure 2 Schematic of error removal strategies using MutS mismatch-binding protein. Assembled gene segments are denatured and re–annealed to form heteroduplexes where errors are revealed as mismatches between correct (blue) and mutant (red) sequences. ...

For error-rich sequences, an alternative MutS-based error-correction strategy is called “consensus shuffling” [39, 40] (Fig. 2b). Re-annealed polynucleotide products are first cleaved by a mixture of restriction endonucleases and the resulting overlapping short fragments are then subjected to MutS column filtration. Short, mismatch-containing pieces are captured by immobilized Taq MutS, while error-free fragments are eluted and re-assembled into full-length constructs by assembly PCR . Two iterations of consensus shuffling improve the error rate 3.5- to 4.3-fold to 1 error per 3.5kb [39].

Consensus shuffling is a mechanism that resembles DNA shuffling where several iterations can be made until the consensus sequences are concentrated enough to far outnumber other species. Consensus shuffling provides several advantages over direct MutS filtration of full-length sequences: i) it is more effective on longer DNA constructs where perfect hybridization after re-assortment is rare; ii) it tolerates more errors in the starting material; iii) the removal of the entire DNA heteroduplex is not necessary. Importantly, fragmentation is ideally performed with type IIS restriction endonucleases that cut outside of the recognition sites. In this way, distinct ends can be generated by the same restriction endonuclease so as to minimize nonspecific hybridization of cut fragments after error-filtration. However, despite the merit of the idea, consensus shuffling requires extraneous use of endonucleases and the reaction conditions may still need to be improved to achieve better outcome [39] Error-correction using mismatch-cleaving enzymes

Mismatch-cleaving enzymes here refer to a group of mismatch-specific endonucleases that recognize and cleave at or near mismatch sites in DNA heteroduplexes. The family embraces a variety of members including but not limited to: i) resolvases, such as phage T4 endonuclease VII [41], T7 endonuclease I [42] and E.coli endonuclease V [43]; ii) mismatch repair endonuclease MutH [38]; iii) single-strand specific nucleases, such as S1 nuclease from Aspergillus oryzae, P1 nuclease from Penicillium citrinum, mung bean nuclease, and CEL nuclease from celery [44, 45]. The ability of these endonucleases to cleave heteroduplex DNA at the mismatch sites has lead to their extensive applications as probes for mutation and polymorphism detection [44, 46–50]. The mismatch-cleaving activity also makes them very useful in error removal for gene synthesis and several different assays were developed utilizing this activity.

Resolvases

T7 endonuclase I was used in an early report:after assembly and amplification of a target DNA, the purified product was denatured, re-annealed and treated with T7 endonuclease I. Afterwards, the pool was run on a gel and the remaining intact band was excised and extracted, while the cleaved fragments were discarded [51]. This way of enriching error-free sequences seems to work for high-quality synthetic products where correct sequences out number mutants.

The efficacy of various resolvases was systematically explored to recognize and cleave single-base mismatches using a different error-removal assay, which is applicable for low-quality sequences. Three resolvases, T4 endonuclease VII, T7 endonuclease I and E.coli endonuclease V were tested respectively in the process of ligation-based gene synthesis and functional cloning of bacterial chloramphenicol-acetyltransferase (cat) gene. After treatment with T4 endo VII and E.coli endo V, the fraction of “functional clones” increased over 10-fold, and sequence analysis revealed an over 4-fold reduction of error-rate. T7 endonuclease I was not fully characterized in this setting [32]. Instead of using gel purification, it was demonstrated that following mismatch-cleavage, an additional 3’to 5’ exonuclease treatment to remove single-stranded overhangs was essential for error removal, either by adding E.coli exonuclease I or using the intrinsic 3’to 5’ exonuclease activity of proof-reading DNA polymerases (Fig. 3). The processed DNA fragments were reassembled into full-length products by assembly PCR. Figure 3 Figure 3 Schematic diagram showing the principal of error removal using mismatch-cleaving enzymes. The re-annealed heteroduplexes are cleaved in both strands by mismatch-specific enzymes 2–5bp downstream of the mismatches, generating stagger ends that ...

Another method, called circular assembly amplification, combines exonuclease and endonuclease treatments to improve gene synthesis quality [52]. In this method, the overlapping and complementary oligonucleotides are first ligated into circular molecules under stringent conditions. An exonuclease is added to degrade any un-circularized polynucleotides. The circular DNA constructs are then treated with E. coli endonuclease V, which linearizes and degrades the mismatch-containing circles in concert with the exonuclease. By using the above three tiers of selections, the method is able to reduce error-rate by at least 7-fold compared to the conventional PCA assembly protocol [52].

A problem with phage resolvase-based mismatch cleavage is preferential cleavage of certain types of mis-matches. For instance, T7 Endonuclease I has been reported to fail to achieve quantitative cleavage for selected mis-pairs [32]. In addition, the degree of specificity of T4-endonuclease VII has been found to be highly dependent on the length of the substrate and the sequence surrounding the mismatch sites [53, 54]. This enzymology limitation is intrinsic to these resolvases because they naturally function to recognized holiday junctions rather than mismatches in a heteroduplex [55]. Their potency and efficacy of mismatch cleavage are therefore compromised by both low sensitivity and high undesired background. Future work to optimize their substrate specificity or use enzyme combinations may potentially produce better results.

MutHLS

E. coli proteins MutH, MutL and MutS constitute a bacterial mismatch repair mechanism: MutS detects and binds to mispaired bases and small single-strand loops, MutL couples the MutH endonuclease to the MutS bound mutant sites, leading to incision of the unmethylated strand of a hemi-methylated dsDNA. The error containing staggered ends are trimmed by a helicase and an exonuclease, and the resulting gap is filled by a DNA polymerase and a ligase [5]. MutHLS can be used to remove polymerase-produced mutant sequences in PCR products [38]. Treating re-annealed PCR products with MutHLS allows MutS to bind to the mismatches and create incisions on both unmethylated strands at d(GATC) in the vicinity of the mismatch sites. The cleaved heteroduplexes are then removed by gel electrophoresis. Mismatch removal using E. coli MutHLS reduces the error rate by one order of magnitude and that is effective to G-T, A-C, G-G, A-A and small insertion/deletion mis-pairs, while less effective for other type of mismatches [38].

Single-strand specific nucleases

Single-strand specific nucleases are a family of multifunctional enzymes that are present ubiquitously and display a wide variety of characteristics. Although more than 30 single-strand nucleases have been isolated from various sources, only a few have been thoroughly characterized. The most widely used single-strand nucleases are almost exclusively extracellular glycoproteins, including S1 and P1 nucleases from fungi, the mung bean nuclease and the CEL nuclease from plant. These nucleases vary drastically in the degree of specificity towards different types of mismatches. For instance, S1 and P1 nucleases appear to be strongly specific to AT-rich regions [55]; S1 nuclease seems incapable of recognizing single base mismatches [56]; and quantitative single nucleotide mismatch cleavage by mung bean nuclease occurs only at pH 6–6.5 [57]. Moreover, contradictory results have been reported regarding the use of S1, P1 and mung bean nucleases in detecting different types of single nucleotide polymorphism (SNP) in heteroduplex DNA [57]. The incomplete capability of these single strand nucleases to identify all possible types of mismatches greatly discourages their general use in synthetic gene error correction.

CEL endonuclease is an ortholog of the S1 nucleases isolated from celery [45, 58]. The uniqueness of CEL endonuclease is that it is the first eukaryotic endonuclease known to cleave DNA with high specificity towards all types of base mismatches and DNA distortions at neutral pH. Following the discovery of CEL nuclease, the techniques for high-throughput mutation detection have significantly improved, and CEL has become the most commonly used enzyme in TILLING (targeting-induced local lesions in genomes) [59–62], polymorphism analysis [63, 64] and disease diagnosis [58, 63–68].

CEL endonuclease is a mannosyl glycoprotein that nicks a DNA strand at the 3’end of the base-substitution mismatch and DNA distortion. Prolonged incubation and a high enzyme to substrate ratio facilities double-stranded cleavage by CEL at opposite phosphodiester bonds 3’ to the mismatch, generating i) two single-base 3’ overhangs in the case of base-substitutions or ii) a short stagger end in the case of insertion/deletion [45, 58]. The commercialization of CEL endonuclease as Surveyor™ nuclease by Transgenomic, Inc (Omaha, NE, USA) has led to its extensive applications in mutation detection as a simple yet cost-effective method, resulting in the discovery of many novel mutations associated with human genetic disorders, including BRCA1 [45, 58, 69], EGFR [65], HCDC4 [66], p53 [67], mitochondrial [63, 64] and kidney-related [68] genes, etc. The broad substrate specificity and low undesired activity of CEL nucleases make them the most promising candidates for error-correction in synthetic gene assemblies.

Recent studies employing on-chip gene synthesis technology explored the effectiveness of Surveyor™ nuclease in error correction of microchip synthesized genes [25, 70] (Fig. 4). The incorporation of a 60-minute digestion step with Surveyor™ nuclease followed by PCR assembly with Phusion DNA polymerase was able to reduce the error rate of the synthetic gene products from 1 error per 526 bp to 1 error per 3,883 bp. Two iterations of enzymatic error cleavage reactions further reduced the error rate to 1 error per 8,701bp, representing an over 16-fold error reduction [70]. Selective sub-pool amplification of chip-synthesized oligonucleotides achieve an error-rate of 1 error per 7,017bp after enzymatic error correction using ErrASE kit, a commercially available CEL-based enzyme cocktail that corrects mismatches [71]in a similar fashion to [25]. Figure 4 Figure 4 Schematic diagram of error correction strategy using CEL mismatch-specific endonuclease. Multiple CEL error-correction cycles maybe integrated into a gene synthesis process. Each cycle consists of four steps: 1) Re-annealing of assembled gene constructs ...

The combined actions of mismatch cleavage by CEL nuclease and proof-reading by Phusion DNA polymerase makes the procedure particularly robust and suitable for correcting error-rich synthetic sequences. Re-annealing of low-quality synthetic sequences often yield little error-free duplexes that can survive the mismatch-binding filtrationor mismatch-cleavage. Rather than discarding all error-containing heteroduplexes, CEL nuclease makes double-stranded cuts next to the mismatches. The mismatch bases are then chewed away by the exonuclease activity of the proof-reading DNA polymerase. The resulting error-free fragments are reassembled into full-length gene constructs by assembly PCR (Fig. 4). This method enables error elimination at the single-base level, while salvaging a majority of the correct portions of the sequences. More importantly, the “inspection-correction-reassemble” cycle can be iterated multiple times until products with desired purity are obtained.