Zinc Finger Nuclease (ZFN) led Editing – CRISPR and Genomic Editing (Part 2)

Hello All!

This will be the 2nd part of a 10+ part post series summarizing current research on Genome Editing and CRISPR. After that I will be moving onto its applications in various diseases such as Cancer. The first few posts will be my summary of research articles that came out in the past few years to get readers caught up (its recommended that readers are somewhat learned in Biology) to present in regards to this technology. I will explain this to the best of my ability and if I make any errors, please correct me by commenting below.

Vocabulary to know:

Restriction Enzyme – an enzyme that helps split or cleave DNA at specific receptor sites. They are mainly found in the Phylum Bacteria and the Phylum Archaea

Homologous – having the same structural features and pattern of genes, the homologous DNA is made through the DNA replication process

S phase – The phase of the cell cycle when DNA replicates

G2 phase – The phase of the cell cycle following the S phase where the cell prepares for division (mitosis or meiosis)

frameshift – A mutation in DNA caused by either the insertion or deletion of the nucleotide. These are dangerous because amino acids are coded for in pairs of 3 called codons and a frameshift can throw off the protein.

Transcription Factors – Specialized proteins that when attached to DNA, can regulate nearby genes (can turn on or off)

Zinc Finger Nucleases:

Last time, we examined the technologies/methods before advanced gene editing technology. Today, we will look at Zinc Finger Nucleases (ZFN). ZFNs are transcription factors attached to the FOK1 restriction enzyme and can artificially be made to target specified sequences in large genomes.

ZFNs recognize the portion to excise through a sequence of DNA on both the forward and  reverse strand. Then, the ZFN will bind to the sequences on both sides allowing a bacterial restriction enzyme called FOK1 to cleave the DNA. This leaves both strands of the DNA cut but to prevent damage to the cell, the DNA will need to be recombined somehow. There are two natural processes for this: homologous recombination and non homologous end joining. JCI72992.f1

In homologous recombination(HDR),  a homologous strand to the excised strands helps the excised strands replicate its DNA back. This can only happen when the cell has a sister chromatid with homologous pairs available. That means this is only viable in the late S stage or the G2 phase.

The other method is non homologous end joining (NHEJ). In NHEJ, the split ends just rejoin with the help of a special enzyme called DNA ligase. Sometimes the NHEJ process works just as well as recombination but in a lot of scenarios the NHEJ process is flawed and results in frameshifts making the gene faulty and thereby changing the expression of the DNA.

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-Sai Rachakonda,

Co-President of STEMTalksNC

https://www.linkedin.com/in/sai-harshith-rachakonda-674941149/

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