Editing Protein engineering (section)

=== Sexual methods ===
Sexual methods of directed evolution involve ''in vitro'' recombination which mimic natural ''in vivo'' recombination. Generally these techniques require high [[sequence homology]] between parental sequences. These techniques are often used to recombine two different parental genes, and these methods do create cross overs between these genes.<ref name=PoluriBook/>{{page needed|date=May 2017}}

==== ''In vitro'' homologous recombination ====
Homologous recombination can be categorized as either ''in vivo'' or ''in vitro. In vitro'' homologous recombination mimics natural ''in vivo'' recombination. These ''in vitro'' recombination methods require high sequence homology between parental sequences. These techniques exploit the natural diversity in parental genes by recombining them to yield chimeric genes. The resulting chimera show a blend of parental characteristics.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====DNA shuffling====
This ''in vitro'' technique was one of the first techniques in the era of recombination. It begins with the digestion of homologous parental genes into small fragments by DNase1. These small fragments are then purified from undigested parental genes. Purified fragments are then reassembled using primer-less PCR. This PCR involves homologous fragments from different parental genes priming for each other, resulting in chimeric DNA. The chimeric DNA of parental size is then amplified using end terminal primers in regular PCR.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Random priming ''in vitro'' recombination (RPR)====
This ''in vitro'' homologous recombination method begins with the synthesis of many short gene fragments exhibiting point mutations using random sequence primers. These fragments are reassembled to full length parental genes using primer-less PCR. These reassembled sequences are then amplified using PCR and subjected to further selection processes. This method is advantageous relative to DNA shuffling because there is no use of DNase1, thus there is no bias for recombination next to a pyrimidine nucleotide. This method is also advantageous due to its use of synthetic random primers which are uniform in length, and lack biases. Finally this method is independent of the length of DNA template sequence, and requires a small amount of parental DNA.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Truncated metagenomic gene-specific PCR====
This method generates chimeric genes directly from metagenomic samples. It begins with isolation of the desired gene by functional screening from metagenomic DNA sample. Next, specific primers are designed and used to amplify the homologous genes from different environmental samples. Finally, chimeric libraries are generated to retrieve the desired functional clones by shuffling these amplified homologous genes.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Staggered extension process (StEP)====
This ''in vitro'' method is based on template switching to generate chimeric genes. This PCR based method begins with an initial denaturation of the template, followed by annealing of primers and a short extension time. All subsequent cycle generate annealing between the short fragments generated in previous cycles and different parts of the template. These short fragments and the templates anneal together based on sequence complementarity. This process of fragments annealing template DNA is known as template switching. These annealed fragments will then serve as primers for further extension. This method is carried out until the parental length chimeric gene sequence is obtained. Execution of this method only requires flanking primers to begin. There is also no need for Dnase1 enzyme.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Random chimeragenesis on transient templates (RACHITT)====
This method has been shown to generate chimeric gene libraries with an average of 14 crossovers per chimeric gene. It begins by aligning fragments from a parental top strand onto the bottom strand of a uracil containing template from a homologous gene. 5' and 3' overhang flaps are cleaved and gaps are filled by the exonuclease and endonuclease activities of Pfu and taq DNA polymerases. The uracil containing template is then removed from the heteroduplex by treatment with a uracil DNA glcosylase, followed by further amplification using PCR. This method is advantageous because it generates chimeras with relatively high crossover frequency. However it is somewhat limited due to the complexity and the need for generation of single stranded DNA and uracil containing single stranded template DNA.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Synthetic shuffling====
Shuffling of synthetic degenerate oligonucleotides adds flexibility to shuffling methods, since oligonucleotides containing optimal codons and beneficial mutations can be included.<ref name=PoluriBook/>{{page needed|date=May 2017}}

==== ''In vivo'' Homologous Recombination ====
Cloning performed in yeast involves PCR dependent reassembly of fragmented expression vectors. These reassembled vectors are then introduced to, and cloned in yeast. Using yeast to clone the vector avoids toxicity and counter-selection that would be introduced by ligation and propagation in E. coli.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Mutagenic organized recombination process by homologous ''in vivo'' grouping (MORPHING)====
This method introduces mutations into specific regions of genes while leaving other parts intact by utilizing the high frequency of homologous recombination in yeast.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Phage-assisted continuous evolution (PACE)====
{{main|Phage-assisted continuous evolution}}
This method utilizes a bacteriophage with a modified life cycle to transfer evolving genes from host to host. The phage's life cycle is designed in such a way that the transfer is correlated with the activity of interest from the enzyme. This method is advantageous because it requires minimal human intervention for the continuous evolution of the gene.<ref name=PoluriBook/>