Researchers used single-molecule imaging in order to compare genome-editing tools CRISPR-Cas9 and TALEN – revealing that TALEN is up to five times more efficient.
Both CRISPR-Cas9 and transcription activator-like effector nuclease (TALEN) are programmable DNA search engines that identify genomic sequences for target-specific editing. Although they both can recognise a custom genetic sequence, they have strikingly different mechanisms of target-site binding. For example, researchers can programme Cas9 to find a specific sequence upstream of a PAM (protospacer adjacent motif) through designing a single guide RNA. Whereas the DNA-binding domain of a TALEN consists of a tandem array of 33–34 amino acid-long customisable monomers.
While in vitro studies have shown that TALEs (nuclease-free analogues of TALENs) use a ‘molecular zip-line’ mechanism for target-site search along DNA, it is not yet known how they manoeuvre in vivo. In addition, previous studies have presented conflicting evidence on the search mechanism of CRISPR-Cas9. Understanding the underlying search mechanisms of genome-editing proteins within the context of cellular chromatin environments is critical for selective recognition of target sites in gene editing.
CRISPR-Cas9 vs TALEN
In this study, published in Nature Communications, using single-molecule fluorescence microscopy, researchers were able to directly observe how the two genome-editing tools perform in living mammalian cells. Moreover, fluorescent-labelled tags enabled the team to measure how long it took these proteins to move along the DNA and then detect and cut target sites.
These analyses revealed that Cas9 is less efficient in heterochromatin than TALEN as Cas9 becomes hindered by local searches on non-specific sites within these regions. Additionally, they found up to a five-fold increase in editing efficiency for TALEN compared to Cas9 in the constrained heterochromatin regions of the genome.
These findings will pave the way for improved approaches for targeting various parts of the genome. This is particularly important for disorders, such as Fragile X syndrome, sickle cell anaemia and beta-thalassemia, where mutations within heterochromatin cause disease.
Image credit: By Teka77 – canva.com