By Dr Tomislav Meštrović, MD, PhD
Gene-editing techniques based on synthetic nucleases and transcription factors have enabled the targeted modification of gene sequence and expression. They have been used to directly targeted gene addition for therapeutic purposes, knock out genes associated with diseases and correct pathogenic mutations.
Currently there are four major classes of engineered nucleases employed for genome editing purposes: zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), engineered meganucleases derived from mobile genetic elements of microbial origin, and the CRISPR system with the RNA-guided Cas9 endonuclease.
Comparison with other techniques
ZFN are the oldest and most established of the aforementioned engineered DNA-binding proteins, most habitually based on the FokI restriction enzyme fused to a zinc finger DNA-binding domain engineered to target a specific DNA sequence. Multiple methods are available to researchers for selecting and designing ZFN targeted to new and desired sites.
TALENs are naturally occurring proteins derived from the bacterial genus Xanthomonas that are similar to ZFNs, as the activity is again through the FokI. Each DNA-binding domain can recognize a different, single DNA base, thus a combination of different TALENs can (in practice) be used to target any specific sequence on the genome.
Both ZFN and TALENs are modular proteins that interact with the major groove of the double helix structure of DNA in order to recognize specific base pairs. Each zinc finger module binds with a nucleotide triplet, whereas TALEN subunits interact with single base pairs. We have ZFN available to target practically all possible nucleotide triplets, albeit individual zinc fingers can display context-dependent effects.
Meganucleases of microorganisms have naturally long recognition sequences (larger than 14 base pairs), and various meganuclease variants have been generated by protein engineering to cover a myriad of unique sequence combinations. Moreover, meganucleases have been shown to cause less toxicity in cells when compared to ZFN and TALENs.
The latest development in this type of technology is the CRISPR/Cas9 system, which are RNA-based defense mechanisms of bacteria and archaea that identify and eliminate alien DNA from attacking bacteriophages and plasmids. One of their main features is Cas9 endonuclease aimed to cleave a target sequence using a guide RNA (shortened as gRNA).
Specificities of CRISPR/Cas RNA-guided DNA targeting
In CRISPR system, the aforementioned Cas9 protein is directed towards the target site using gRNA, which consists of a 20-base pair protospacer that attaches to the complementary strand of the target sequence, as well as a constant region interacting with the Cas9 protein.
The target sequence must be promptly followed by a protospacer-adjacent motif (usually shortened as PAM) for recognition by Cas9 protein. Thus effective DNA targeting by CRISPR/Cas9 is accomplished by choosing a protospacer that is complementary to the relevant genomic sequence.
As specificity is dictated by DNA complementarity (without the need for multistep protein engineering), the CRISPR/Cas technology has entered the picture as the faster, more straightforward and affordable way for genome-editing in comparison to traditional ZFN and TALENs approaches.
Furthermore, systems based on Cas9 have shown the propensity to target heterochromatin sequences, targeting DNase-inaccessible locations and cleaving at highly methylated regions; this fact (in addition to the flexibility offered by multiple variants of Cas9 for selection of targets) lends this approach phenomenal versatility for genome engineering.
Last Updated: Jan 13, 2016