SynBio2: Reading, Writing and Editing DNA and directed evolution. (v1.0)

Before dwelling more deeply into SynBio, we need to examine more broadly the four key tools that are used. To read DNA. To write DNA. To Edit DNA. I will also touch on a key tool used called directed evolution. 

Directed evolution is an important tool for SynBio. It mimics the Darwinian evolution process in the test tube. In a typical directed evolution experiment, the gene encoding a macromolecule of interest is randomized and expressed in a suitable host. Appropriate screening or selection methods are then used to identify mutants that have particular properties of interest, such as binding to a specific small molecule or catalyzing a desired chemical reaction. Through iterative cycles of mutagenesis and amplification of selected mutants, beneficial desired mutations accumulate as in genuine Darwinian evolution but on a vastly shorter time scale. In this way, populations of macromolecules may be deliberately evolved toward useful synthetic and therapeutic properties. 

The ability to read DNA (called DNA sequencing which is deciphering the ordered sequence of nucleotides - Adenine, Cytosine, Guanine and Thymine - in a DNA strand) and the corresponding RNA (uses Uracil instead of Thymine) is vital and today is commonplace and done very rapidly (3 to 12 weeks) and costs less than $1000. In comparison the Human genome project in the 1990's took 13 years and cost $3 billion. The ability to read DNA has also made the ability to edit DNA far more efficient to SynBio scientists. Reading gene sequences lets scientists evaluate how genes change, either through normal mutation and evolution or directed evolution experiments in SynBio.  SynBio scientists also are constantly reading natural sequences of DNA to look for useful or harmful properties. For example, in Chromosome 17, the mutations in the BRCA1 gene are associated with about 40 to 50% of hereditary breast cancer. SynBio scientists also are constantly verifying their own experiments to see if their edits took hold. DNA is scattered everywhere, and the field of Metagenomics focuses on reading DNA directly from our environment to understand our environment and health risks better (example wastewater sampling). DNA sequencing is also the foundation for personalized medicine and early detection of risks of genetic diseases. 

DNA sequencing technology was first invented in the 1970's by Frederick Sanger for which he received a Nobel prize in the 80's. The development of florescence-based sequencing methods in the 90's with a DNA sequencer vastly speeded it up and made it much easier. The sample is first amplified (copied many times) using the polymerase chain reaction (PCR). This produces a much larger sample to work with. Second step is to Introduce dideoxynucleosides that fragments it. Last step is to analyze the fluorescent labels on the DNA fragments to determine the sequence. Many other advancements were subsequently made including NGS, third generation sequencing, Shotgun sequencing and Metagenomic sequencing.

Bottom-up biology in SynBio focuses on piecing together individual parts to get higher function. The writing or printing of DNA is key to synBio and is called artificial DNA synthesis. This synthesis is done without a template but de novo (from scratch anew). It allows virtually any DNA sequence to be synthesized in the laboratory. The first step in artificial DNA synthesis is design on a computer. The second step is printing in a DNA synthesis machine. Short fragments called oligonucleotides with typically less than 200 base pairs are first synthesized. The next step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Further details are beyond the scope of this blog. The efficiency, speed and cost effectiveness with which this can be done will govern how much impact SynBio will have. Also minimizing errors from occurring is crucial. The chance of errors increases with length. 

Synthesis of the first complete gene, a yeast tRNA was demonstrated by Har Gobind Khorana and coworkers in 1972. Synthesis of the first peptide and protein coding genes was performed in the laboratories of Herbert Boyer in 1977 and Alexander Markham in 1981 respectively. More recently, artificial gene synthesis methods have been developed that will allow the assembly of entire chromosomes and genomes. The first synthetic yeast chromosome was synthesized in 2014, and an entire functional bacterial chromosome has also been synthesized. 

Some 100,000 Black people in the U.S. are afflicted with the sickle cell disease which is a genetic disease. A treatment is to have the DNA modified with CRISPR-Cas9 gene editing to knock out a genetic mutation that causes the disease. After six years of work, in 2021, that experimental treatment has now been approved for clinical trials by the U.S. Food and Drug Administration, enabling the first tests in humans of a CRISPR-based therapy to directly correct the mutation in the beta-globin gene responsible for sickle cell disease. Beta-globin is one of the proteins in the hemoglobin complex responsible for carrying oxygen throughout the body.

With CRISPR-Cas9, scientists can now edit genes with relative ease and insert it into the subject. It works across species and cell types. CRISPR-Cas9 was invented in 2012 by Charpentier and Doudna and the creators were awarded a Nobel prize for the work. It has extremely vast potential and has revolutionized many areas. The details of how it works are beyond the scope of this blog.