gene editing

What Genome Editing Was Like Before CRISPR

CRISPR is a genome engineering tool that has been gaining popularity in recent years. However, not a lot of people know that prior to CRISPR, scientists have been using zinc-finger nucleases (ZFNs) to edit DNA. ZFN is the pioneer in the field of gene editing. A zinc finger DNA-binding domain and a restriction endonuclease domain are involved in engineering an enzyme using the ZFN method. The first domain is designed to target and bind to specific sequences of DNA while the latter cleaves the DNA at the desired site.

genome engineering

A look at other genome engineering tools

ZFNs may have represented the first breakthrough in site-specific genome engineering but they have several limitations. They exhibit off-target effects, are expensive, and time-consuming to engineer. Their application is also only limited to only one genomic edit at a time.

Transcription activator-like effector nucleases (TALENs) entered the gene editing scene years after ZFNs made their debut. Similar to ZFN, the TALENs method utilizes engineered enzymes containing a DNA-binding domain and a separate DNA-cleaving domain. The advantage that TALENs have over ZFNs is the flexibility of their DNA-binding domains to target a wider range of sequences. However, TALENs are too costly to produce. 

Adeno-associated virus (AAV) is a non-pathogenic virus, meaning one that does not cause diseases, harm, or death to another organism,  that infects mammalian cells at all stages of the cell cycle and incorporates with the host genome at predictable sites. This versatile viral vector technology has unique biological and biophysical properties which can be engineered for very specific functionality in gene therapy applications and used in a variety of clinical applications in multiple diseases.

Useful as an additional genome editing technique by means of engineering restriction enzymes in concert with recombinant adeno-associated viruses (rAAVs), the AAV genome can be modified to target specific sequences in the host genome and integrate desired modifications. However, the approach has limitations. One, the vectors are difficult to produce and can only accommodate a small amount of genetic material. 

genome engineering

Almost there

The above-mentioned gene editing technologies all came with limitations. ZFNs and TALENS require complex protein-DNA interactions making them challenging to design and manipulate while AAV vectors have limited applications and small packaging capacity, again giving researchers a hard time working with them. 

Then came CRISPR. CRISPR is a gene editing tool that stands out among all because it relies on well-understood interactions between DNA and RNA, offering a far simpler way of editing genes and thus completely changing the face of genomic engineering.

 

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