Gene Editing

Genome Editing

Zinc Finger Nucleases (ZFNs)

• Contain a zinc finger (DNA-binding) domain and a DNA cleavage domain.
• Typically there are 3-9 zinc fingers that each can be engineered to recognize a 3 bp sequence.
• The cleavage domain that is a nonspecific nuclease (FokI) that must work as a dimer.

Transcription Activator-Like Effector Nucleases (TALENs)

• Consist of the TAL effector DNA-binding domain and a DNA cleavage domain.
• The TALE DNA binding domain has a conserved 33 -34 amino acid region that has a variable 12th and 13th position.
• This allows it to bind specifically to the DNA.
• Each TALE binds a single nucleotide.
• The cleavage domain has the nonspecific nuclease (FokI).

CRISPR-Cas9

• Derived form bacterial defense mechanisms.
• Clustered Regularly InterSpaced Palendromic Repeats (CRISPR).
• A single-guide RNA (sgRNA) is often used which has a target sequence and a Cas9 interacting sequence.
• The target sequence is preceded by a PAM sequence.
• PAM = protospacer adjacent motif.
• The sgRNA binds specifically to a region of the genome and Cas9 makes a double stranded break.

CRISPR

1. Bacteria cleaves the invading phage DNA and adds the segments into the CRISPR array.
2. Transcription of the array contains mRNA with CRISPR repeats and invading DNA.
3. Repeat sequence binds to tracrRNA which provides a scaffold for Cas protein.
4. When the bacteria gets infected again with that phage, the complex base pairs with the phage DNA.
5. Cas cleaves the phage DNA.

• Cas9 - engineered version of the Cas endonuclease.
• Guide RNA - engineered to bind to Cas9 and to a specific site on genomic DNA (site of choice).
• This site can be any gene the researcher chooses to inactivate or replace!
• When Cas9 and the guide RNA are both expressed in cells, Cas9 will cleave the DNA at the targeted site.
• The DNA will often repair itself through Non-Homologous End Joining, resulting in the loss of a few base pairs.
• Can cause a deletion or frameshift which inactivates the gene.
• Genes can also be introduced if the cell repairs itself through Homology Directed Repair (HDR) mechanism.
• Add segments that match the sequences flanking the cleavage site.
• When the DNA is cleaved, homologous recombination will insert the donor DNA sequence into the cleavage site during DNA repair.