Genome editing or genome engineering refers to the ability to alter an organism’s DNA by adding, deleting, or substituting nucleotide sequences at specific sites within the DNA. Unlike initial genetic editing technologies, genome editing allows the precise and site-specific alterations in an organism’s genome. The recent developments of versatile genome-editing technologies have revolutionised human genome research. It is now possible to understand the role of a single-gene product in a disease and develop gene-based therapies.
The availability of nucleases (bacterial or engineered) allows researchers to practically edit any genomic sequence both rapidly and economically. It also allows the creation of isogenic cell lines and animal models for the study of various diseases. In the past decade, nuclease-based genome editing technologies have developed rapidly and found wide application in biotechnology and biomedical research.
CRISPR/Cas, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) are the three foundational genome editing technologies that are extensively researched and well-documented. ZFNs are fused nucleases containing an array of zinc finger domains and tandem domains capable of binding to the target DNA. Thus, ZFNs are capable of binding and cleaving DNA at specific site. Similar to ZFNs, TALENs rely on the use of TALE repeat arrays for recognising and cleaving DNA at a particular site.
However, both ZFN and TALEN technologies have limitations. For example, zinc finger domains are complex and thus difficult to engineer in laboratories. ZFN can bind to the target DNA after every 200 base pairs (bp). This limits the potential of ZFN to cleave the DNA at any specific site. Additionally, ZFN is associated with high off-target effects that can generate unintended mutations, thus leading to undesired side effects. Although compared to zinc finger domains, TALE arrays are relatively easier to engineer in laboratories, TALEN is also associated with a high off-target effect. Furthermore, TALE arrays due to their larger size (3 kb) compared to ZFNs (1kb) are difficult to deliver within cells for genome editing.
CRISPR/Cas technology relies on the functioning of Cas nuclease and complementary binding of the single guide RNA (sgRNA) with the protospacer in the target DNA and the presence of protospacer adjacent motif (PAM). Thus, by modifying the sequence of the sgRNA, the CRISPR/Cas system can be adapted to target any DNA sequence without having the need to engineer the Cas endonuclease. Compared to ZFNs and TALENs, the CRISPR/Cas system is cheaper and has higher specificity and efficiency in genome editing, thus making CRISPR/Cas the preferable genome editing technology.