CRISPR-Cas9 has made waves in the biomedical world as a revolutionary gene editing tool, even garnering a 2020 Nobel Prize in chemistry. But it has its limitations.
A research team from the University of Illinois at Urbana-Champaign (UIUC) showed that another gene editing technique called TALEN is up to five times more efficient than CRISPR-Cas9 in a highly compact form of DNA called heterochromatin, according to results published in Nature Communications.
The findings point to TALEN as a better option for the engineering of some hard-to-edit genomic regions, which could be applicable to both research and therapies, the scientists argued. Genetic defects in heterochromatin can cause such diseases as sickle cell anemia, beta thalassemia and fragile X syndrome.
In the CRISPR system, a tailored, single-guide RNA carries DNA-cutting enzymes such as Cas9 to target sites. TALEN also scans DNA to find and target specific genes. Molecules called transcription activator-like effectors, or TALEs, are fused with the DNA scissors, Fok1. On TALE, the highly variable amino acid positions called repeat variable diresidues, or RVDs, act as guides that can recognize specific nucleotides for binding.
But scientists don’t fully understand how TALEs and CRISPR-Cas9 navigate the complex environment of the cell nucleus and search for their target sites in living beings.
For the new study, the UIUC scientists used single-molecule fluorescence microscopy to observe the search dynamics of TALE and a Cas9 mutant. The fluorescent molecule allowed them to measure the time CRISPR and TALEN took to get to their target sites.
The Cas9 protein spent more time than TALE did on both nonspecific sites and local search, the team found. In heterochromatin, both TALE and Cas9 appeared to encounter a considerably constricted search space, the team found. But in at least at one highly compact heterochromatin located in chromosome 16, TALE demonstrated significantly faster search dynamics compared to Cas9.
To evaluate the functional implications of the different search behaviors, the team constructed a series of TALENs and Cas9 guide RNA variants to see how they would edit DNA sequences in highly repressed heterochromatin regions. In 11 out of 12 loci, TALENs showed similar or higher editing activity compared with Cas9.
In some cases, TALENs were over five times more efficient in editing than Cas9-gRNA, the team found.
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TALEN is one of several alternate gene editing technologies under development. A Columbia University team used a “jumping gene,” or transposone, from a bacterium called Vibrio cholerae to insert DNA sequences at targeted locations, for example. That system doesn't require the double-strand break that TALEN and CRISPR-Cas9 editing use.
Findings from the UIUC study suggest multiple gene editing tools may be needed, the scientists argued. For one thing, Cas9 appeared to be better at editing in euchromatin sites, the team found. Euchromatin is the most transcriptionally active portion of the genome within the cell nucleus. The researchers proposed that Cas9’s enhanced local search ability is beneficial in a less hindered environment but counteractive in more compact regions.
“Overall, these results serve as a guide in selecting genome-editing proteins for the engineering of hard-to-edit heterochromatin regions of mammalian cells for general as well as therapeutic purposes,” the researchers wrote in the study.