Evolutionary biologists sometimes speak of the mode and tempo of evolution, where the mode encompasses how a molecule or organism moves along a fitness landscape, and the tempo is of course the speed at which the landscape is traversed.  Synthetic evolution is no less subject to these considerations, and as with other aspects of this field, there are remarkable new opportunities for altering both mode and tempo. 

Amusingly, the mode and tempo of the field, as well as the outcomes available via the tools and approaches being developed, are in flux.  Even since the acceptance of our piece there have been several major advances in how synthetic evolution can be attempted.  Continuing the trend of using the CRISPR revolution to create new site-directed tools, two groups (Feng Zhang’s at MIT, and Samuel Sternberg’s at Columbia) have melded Tn7 integration functionality with RNA-directed binding to DNA in order to generate fusion proteins that can direct the efficient and targeted integration of large (several kilobase) insertions into genomes [1, 2].  This system is reminiscent of ‘tagmentation’ methods for sequencing, but may allow guided genome evolution at scale.  While there are other methods for directing genomic insertions, such as recombineering, and while CRISPR systems have previously been adapted to efficient genome editing (such as the CREATE method), the ability to have large donor fragments inserted at will based on the transformation of donor plasmids should greatly accelerate genome engineering. 

A similar melding of natural functionalities was also apparent in the CRISPEY system [3], which fuses CRISPR gRNAs with the template RNAs for retron reverse transcriptases, thereby allowing the site-specific production of single-stranded DNAs that can presumptively be incorporated into genomic DNA as Okazaki fragments.  While only short edits of the genome are possible, the efficiency is once again extremely high, even relative to other CRISPR systems.  Most importantly, like other retron-based approaches, the CRISPEY system can potentially operate iteratively, opening the way to accelerated continuous evolution.

While it stands to reason that the tempo of evolution will increase as tools that have previously accelerated bacterial evolution (CRISPR, retrons) are adapted to multiple new functions and cell types, how this will impact the mode of evolution is less clear.  Base changes and genomic rearrangements already occur, and while fitness landscapes will be traversed far more quickly, it seems likely they will nonetheless be much the same fitness landscapes that were already available to natural selection.  Into the future, it may be the tools of chemical biology that break the paradigm of natural selection, and more definitively change the mode of evolution.  Novel nucleotide pairs are expanding the genetic alphabets of cells [4], and plans for concomitant expansion of the genetic code are underway [5, 6].  When combined with the previously Herculean, and now more routine, synthetic recoding of entire genomes, the day is not far off when Brave New Organisms with greatly expanded monomer sets, and therefore greatly expanded chemistries, may co-exist with those of us that have canonical nucleotides and amino acids. 

It is entirely unclear how such organisms will evolve.  Even at the most basic level, it is an open question as to how an increased number of nucleotides and amino acids will generally affect nucleic acid and protein folding.  While certainly there will be interesting new folds and functions, there will also be the possibility for new mis-folds.  Even in just thinking about the secondary structures of nucleic acids, it seems likely that the number of available, stable ‘off-target’ secondary structures will scale in some way with the size of the genetic alphabet, relative to a single desired on-target structure.  The information required to appropriately specify an on-target structure with a 6- or 8-letter alphabet is currently unknown, and while it will likely depend on the idiosyncracies of cross-pairing and stacking, it may be greater than for a smaller, 4 letter alphabet.  Should this hypothesis play out in 3 dimensions, and with other biopolymers, expanded genetic codes may merely give us much more intracellular protein aggregation.

That said, nature has previously worked out how to avoid the mis-folds of 4 letter genetic alphabets and 20 amino acid genetic codes, and the accelerated tempo of synthetic evolution may do the same for Brave New Organisms, as well.  Indeed, it seems most likely that this accommodation will occur in stages.  In the present, new nucleotides and amino acids are being introduced sparingly, and their genome- or proteome-wide consequences are largely untested and unknown.  As researchers grow more confident with the tools for introduction, smaller replicons (plasmids, phage) will accommodate the expanded alphabets and codes.  Accommodated genes and circuits can then be moved into organisms with larger genomes, first in unique sectors that will function aside the normal machinery of the cell, perhaps with their own polymerases and ribosomes, creating orthogonal biochemistries.  And finally the true Brave New Organisms, with fully integrated chemistries, will emerge and teach us all over about how life evolves, and whether new chemistries do indeed lead to new modes of evolution.

1.  Klompe, S.E., et al., Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature, 2019.

2.  Strecker, J., et al., RNA-guided DNA insertion with CRISPR-associated transposases. Science, 2019.

3.  Sharon, E., et al., Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing. Cell, 2018. 175(2): p. 544-557 e16.

4.  Malyshev, D.A., et al., A semi-synthetic organism with an expanded genetic alphabet. Nature, 2014. 509(7500): p. 385-8.

5.  Zhang, Y., et al., A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proc Natl Acad Sci U S A, 2017. 114(6): p. 1317-1322.

6.  Dien, V.T., et al., Expansion of the genetic code via expansion of the genetic alphabet. Curr Opin Chem Biol, 2018. 46: p. 196-202.