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Producing custom DNA inside of cells
Innovations in harnessing retrons for biotechnological applications
In university, I would often visit my molecular biology professor in his small office for long discussions about science. In one of these instances, he asked me, “Are you a protein person or a DNA person?”
By this, he was asking me if when I thought about mechanism in biology, I imagined biochemical events such as ligands binding to receptors, or of genes as the causal actors, with exquisite cascades of precisely timed regulatory events.
I, dear reader, am a DNA person1. While I am continually in awe of the recent advances in protein design and modeling, and am excited to see what companies such as Nautilus Biotechnology will do for the field of proteomics, the centrality of DNA for both biology and biotechnology is hard to argue with.
The ability to synthesize or assemble DNA and RNA is absolutely foundational for both biological research and biotechnology. Synthetic DNA is the primary substrate for gene editing, and with COVID-19 we have all learned the importance of synthetic RNA. If you don’t believe me, you might take Elon’s word for it:
This hyperbolic tweet from the person who is simultaneously the richest man in the world and the biggest Twitter troll in the world was met with a collective groan from scientists, and a sarcastic “thanks” conveying a sentiment best captured by xkcd:
But fundamentally, using this type of language to describe the role of DNA in biotechnology is far from a new idea, and has been central to the field of synthetic biology (synbio) since its inception. For example, a classic synbio paper from 2005 describes the “refactoring” of a viral genome2.
This concept is not an analogy. For example, the incredibly talented and creative scientist Seth L. Shipman demonstrated the encoding of a digital movie into bacteria using CRISPR while working as a postdoc with George Church3.
In 2018, Seth Shipman started a lab at the Gladstone Institutes in San Francisco to carry out more research at the intersection of biology and technology. For this installment of The Century of Biology, I am going to highlight a new pre-print from the Shipman Lab entitled “Improved architectures for flexible DNA production using retrons across kingdoms of life”, which describes new improvements for producing DNA inside of cells for biotechnological applications.
So far we have looked at the importance of producing DNA for biotechnology. With this idea in mind, a crucial implementation detail arises: if we want to encode instructions in DNA, how do we make that DNA available to a cell?
Conventionally, the answer has been to produce exogenous DNA outside of a cell, and then deliver it to cells. For the software engineers in the audience, this is like including the instructions as a runtime dependency rather than a build-time dependency. As an engineer, I’ve experienced the overhead of complexity that this can involve. It turns out that this can also be suboptimal for cells, leading to inefficiency in gene editing due to delivery.
An alternative strategy is to produce the desired DNA inside of a cell, using its own native molecular machinery. In this pre-print out of the Shipman Lab led by co-first authors Santiago Lopez and Kate Crawford, the team focuses on optimizing retrons as tool for exogenous DNA production.
What is a retron?
A retron is a genetic element in bacteria that encodes reverse transcription of an RNA sequence into DNA. The architecture of retrons is very minimal, only consisting of “a reverse transcriptase (RT), a non-coding RNA (ncRNA) that is both the primer and template for the reverse transcriptase, and one or more accessory proteins.” Similar to CRISPR, these elements play a role in bacterial defense against viruses. This RT-DNA in the cell can then serve as a template for gene editing. The diagram above illustrates the reverse transcription of the Eco1 retron from ncRNA (pink) into DNA (blue).
While the proof-of-concept for using retrons for gene editing has been successfully shown, the devil is in the details. This work aims to develop a more flexible and optimized framework for producing DNA with retrons across multiple distinct kingdoms in the tree of life.
A critical goal in optimizing retrons for DNA production is to demonstrate that a greater amount of RT-DNA can be produced compared to what would be delivered from synthesized exogenous DNA. The authors tested several ways to modify retrons in order to achieve this goal.
To start, the group synthesized a library of retron variants with modifications in the length of the hairpin stem of the RT-DNA.
They found that only a fairly tight window around the wild-type stem length generated as much RT-DNA, and decided on a window of 12-30 bases for stem length moving forward.
The next modification investigated was the length of the a1/a2 complementarity. The authors describe a1/a2 complementarity as “a region of the ncRNA structure where the 5’ and 3’ ends of the ncRNA fold back upon themselves, which we hypothesize plays a role in initiating reverse transcription.”
The result was exciting:
They demonstrated that increasing a1/a2 length resulted in “the first modification to a retron ncRNA that has been shown to increase RT-DNA yield.” While I found this to be an incredibly exciting result, this turned out to not be the only first in the paper.
In the remainder of the pre-print, the researchers go on to demonstrate “the first demonstration of RT-DNA by a retron in human cells”, and to validate that their retron modifications extend to use in gene editing.
One interesting result is that extending the a1/a2 region actually reduced RT-DNA production in human cells. This is an example of how much room there is for further optimization and exploration of this system.
Thank you for reading this highlight of the new pre-print “Improved architectures for flexible DNA production using retrons across kingdoms of life” from the Shipman Lab at the Gladstone Institutes in San Francisco. If the thought of becoming a molecular tool builder sounds more exciting than optimizing ad revenue, you should consider applying to work with the Shipman Lab! The young group is looking for new members, and this is a chance to get in on the ground floor with a lab doing some of the most exciting and innovative work in synthetic biology.
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This is clearly a simplistic classification, but can be an interesting question to consider. One really exciting protein-centric research program is that of Mohammed AlQuraishi, who just started a new lab at Columbia with the bold vision of simulating a cell with structural resolution by 2050. Such systems-level “structural cell biology” will be incredible to see.
The paper is "Refactoring bacteriophage T7" from the Endy Lab. This is an arbitrary example, and I’m not going to detail the history of synthetic biology, DNA-based programming languages, or the entire field of DNA computing.
The Nature article is “CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria”