In this regard, it is important to consider that virtually all evolution of sex theories only deal with homologous recombination, despite the frequent occurrence of non-homologous recombination in nature. Those recombination methods permit shuffling of disparate gene elements. As its name suggests, epPCR copies the parent genes while introducing mutations as errors, mimicking naturally occurring, imperfect, DNA replication. Most probably, mutation and recombination together constituted the basic evolutionary mechanisms allowing the appearance of RNA variants further selectable and replicable in a progressively successful RNA world [ 85 ]. Two recent evolution experiments with asexual bacterial populations of varying population size [ 89 ] and population structure [ 90 ], suggest that conditions leading to inefficient selection may sometimes provide adaptive benefits, which were ascribed to the ruggedness of the fitness landscapes involved. Crucially also, observed recombination effects can be correlated with the topography of the local fitness landscape to examine the origin of linkage disequilibria underlying observed effects. Iterative rounds of random mutagenesis and selection have been the most frequently used method in directed evolution experiments. Note, however, that finding conditions allowing maximal long-term selection responses is not only relevant for gaining fundamental insights in the evolutionary benefits of recombination, but also for maximizing responses in biotechnology.
As its name suggests, epPCR copies the parent genes while introducing mutations as errors, mimicking naturally occurring, imperfect, DNA replication. Selected properties include the ability to bind to a specific target, the ability to catalyse a desired chemical reaction, or increased stability in specific conditions [ 33 ]. However, rapidly developing microfluidic and millifluidic [ 96 , 97 ] technologies and decreasing sequencing costs hold the promise for much higher throughput fitness assays in the near future. If there is little or no sequence homology e. Presently, relatively small sequence spaces can be investigated, which severely limits the region of the fitness landscape that can be analysed. For example, several in vitro evolution attempts to improve the activity of an RNA polymerase ribozyme [ 72 ] resulted in relatively modest gains [ 73 ]. Another future goal will be to move from the study of a single, monomeric gene with a single function to more complex allosteric proteins and small genetic networks and operons to study the benefit of recombination in increasingly complex, yet also more biologically interesting systems. Only studies which compare the benefits of sex and recombination with asexual evolution, are reported. This allows for the detection of small effects of recombination, as well as a systematic comparison of mutation and recombination-based evolution schemes, mimicking different evolutionary timescales. These observations suggest that recombination is probably older than LUCA last universal common ancestor and hence of life as we know it. In some of these studies, benefits of recombination have been observed. It would be very informative to use the same pipeline we are proposing for comparing the effect of homologous versus non-homologous recombination, in similarly controlled ways e. Those methods were developed with the aim of obtaining: Most experiments are designed to produce improved variants in a continuous search uphill even if some have combined neutral drift and selection [ 88 ], and in vivo experiments have sometimes found long-term adaptive benefits of such regimes [ 89 , 90 ]. In the past 20 years, a broad array of recombination methods have been reported extensively reviewed in [ 34 , 37 ] , such as family shuffling [ 38 ], staggered extension PCR StEP [ 39 ] and RACHITT random chimeragenesis on transient templates [ 40 ]. Also, Liu and co-workers [ 43 ] compared the effectiveness of non-homologous random recombination NRR with that of epPCR in the evolution of a DNA aptamer using identical starting sequences and selection conditions. However, Fisher's theorem assumes infinite population size, and may not generally hold for finite populations, particularly on rugged landscapes. Typically, in directed evolution studies, researchers are only interested in the best variants that are selected and often ignore the evolutionary paths taken by the evolving biomacromolecules. Indeed, there are very few studies that have directly compared asexual and sexual evolution protocols. Inspired by the theoretical advantage of sex in natural populations and in artificial breeding, he intentionally introduced a sexual mechanism in the evolutionary design strategy unintentional recombination can take place by template switching during PCR [ 36 ]. However, the original protocol for DNA shuffling was also mutagenic, and thus the observed advantages may have come from the additional mutations introduced during in vitro recombination. Directed evolution experiments using in vitro recombination protocols. Crucially also, observed recombination effects can be correlated with the topography of the local fitness landscape to examine the origin of linkage disequilibria underlying observed effects. Strong selection was found to result in short-term adaptive benefits, as predicted by Fisher's theorem, but these immediate benefits were traded off against longer-term disadvantages. Benefits for non-homologous recombination have no asexual reference, but may still exist.
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