The past five or so years has been a bit of a roller-coaster for any field that dabbles in genetics. The advancements the scientific community has made in such a short time frame are nothing less than extraordinary and things only seem to be accelerating from here on out. And the single piece of technology that has made all of this possible, that have opened up the doors to the future, is none other than CRISPR.
Though Cas9 should rightfully get most of the credit, all of the other versions of the tool play a role in this achievement as well. Now, the next step in that evolution can be discussed and it, appropriately, has to do with the evolution of genomes themselves.
While modern genetic technologies can allow us to utilize traits from across the kingdoms of life for their maximum useful purpose, that still relies on us finding those traits in the first place. And it could very well end up being that the traits we desire don’t currently exist or exist in a species or population that we haven’t been able to uncover as of yet. We will keep looking, of course, but that may end up being a fruitless endeavor. So, what other options allow us to generate new traits?
If your answer was mutagenesis, then you are completely right. The standard mutation rates seen in the wild already result in many random and exciting combinations of genes expressed as phenotypes. It has been over a century since we learned that we could speed up this rate of change through the use of mutagenic chemicals or focused radiation.
Since the beginning of the genetic revolution several decades back, scientists have tried to create a more advanced version of this mutation-enhancing system. Their efforts largely were restricted to single gene loci in cells under very strict conditions without much variation allowed. So broader usage was mostly out of the question. We’ve gotten more widespread use out of single nucleotide alterations, but those are too constrained in many cases to create the kind of changes desired.
It is DNA polymerases with their ability to synthesize and construct DNA that have clearly been the potential Holy Grail if we could get them to function as we want. Since they are able to do the 12 kinds of substitutions of nucleotides and the deletions that are needed and on a wider scale than any other enzyme. They vary quite significantly in efficiency and speed though, thus research into them includes finding the exact one that would work for gene editing purposes.
Evolution In Your Hand
A research team at UC Berkeley were able to isolate the kinds they wanted to work with, nick-translatable polymerases that are able to act when one of the strands in double-stranded DNA is cut, causing them to displace the nucleotides that are downstream and degrade them. Thus, if such a polymerase was recruited alongside a specialized form of Cas9 that creates nicks, called nCas9, then it could be used to cause targetable mutagenesis within a gene region.
The group decided to call their new tool EvolvR. With it, they are able to control the start site of mutagenesis by altering the specificity of the nCas9. Other features, such as length of the open mutation window, the rate of mutation, and the need for bias in substitution towards certain kinds of nucleotides, can be controlled via the “processivity, fidelity and misincorporation bias” of the precise nickase polymerase they choose to use in the complex.
Their first test with it combined nCas9 with DNA PolI from Escherichia coli and the plasmid with the total complex was tasked with mutating the sequence of a second plasmid over the course of 24 hours. It properly caused mutagenesis within a targeted 17 nucleotide window, meeting the range of 15-20 nucleotide processivity that DNA PolI contains. They confirmed substitutions of all four nucleotide types, though the amount of substitutions was lessened near the 5’ beginning of the region.
Covering Every Experimental Base
The scientists confirmed with a control group of unfused nCas9 and DNA PolI that without such a fused complex, the two were only able to make a single low-level substitution over the same time period. Other control groups, such as using an offtarget guide RNA and just using nCas9 alone, both were unable to cause a single substitution. This means that off-target effects are not a major concern, as the complex will not force mutagenesis in regions that don’t match the guide sequence. However, different polymerase variants can impact this to some extent.
Subsequent tests were able to improve the mutation rate by adding changes to the DNA affinity of the nCas9 so it more quickly dissociates from the target DNA after causing the desired nick. This increased the mutation rate by 8.7-fold over previous experiments. Furthermore, by adding in a thioredoxin-binding domain (TBD) from bacteriophage T7 into the DNA PolI, they were able to enhance its processivity, allowing it to have an editing window 56 nucleotides wide.
An additional experiment allowed them to focus the mutagenesis of EvolvR on a particular phenotype, such as green fluorescent protein (GFP), increasing the prevalence of the trait in the bacterial population. This could allow the tool to have a utility in heightening population ratios for desired traits that only exist in a small percentage of that population. Happily, the tool also doesn’t impact cell growth or cell viability, so there were no observed toxic effects from employing it.
The final experiment they tried was using several guide RNAs at once to see if EvolvR could cause diversification in multiple spread out and distant gene regions. They were successful and showed that EvolvR could be used for expressions that involve several gene sequences across a genome all at once. It can create extended evolutionary mutations against selective pressures, rapidly presenting a population with a means to respond to those pressures.
The Door To Nature’s Method
The tool this team has created can truly be said to have the capacity to evolve a cell toward a wanted path or to see if there are ways to escape from certain pressures that, in nature, might take hundreds, thousands, or even more years to appear. The researchers believe much more work is still required to improve the mutation rate and continue expanding the editing window, along with finding a way to target low-transformation cell types more readily. But it is a massive first accomplishment in extended diversification of nucleotides in a genome and there is likely far more to come.
Photo CCs: Group A Streptococcus Bacteria on Human Neutrophil (8517040030) from Wikimedia Commons