Part 5: Read, Write and Edit

DNA Read

What DNA would you want to sequence (e.g., read) and why?

I would like to sequence the plasmid I modified (pLVX-EF1alpha-POU5F1-7xHis-IRES-mCherry) as a way of experimentally verifying that there were no mutations and that the junctions integrated properly.

In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why?

I would use the “Oxford Nanopore” sequencing method because it would allow me to find accidental recombinations and deletions by being able to sequence the entire plasmid.

DNA Write

What DNA would you want to synthesize (e.g., write) and why?

I would like to synthesize de novo the entire modified POU5F1 (Oct4) cassett, because that way I can perform codon optimization and add the necessary elements (histidine tag and Kozak sequence) in the most efficient way.

What technology or technologies would you use to perform this DNA synthesis and why?

I would use silicon-based DNA synthesis on microchips. Given that the Oct4 cDNA is approximately 1 kb, I really appreciate the ability to synthesize thousands of oligonucleotides in parallel with extremely high accuracy and at a much lower cost than traditional column synthesis.

DNA Edit

What DNA would you want to edit and why?

I would like to edit the genome of the plasmid recipient cells so that they possess a specific locus where the POU5F1 cassette can be inserted without activating oncogenes or disrupting vital genes.

What technology or technologies would you use to perform these DNA edits and why?

I would use Prime Editing because it would allow me to edit a small portion of the genome with considerable precision and turn it into a specific recognition site. Since it uses a Reverse Transcriptase (fused to Cas9 nickase) to write the new sequence directly into the DNA, it works perfectly in neurons or resting fibroblasts.