CRISPR-based enzyme modifiers improve likelihood of inserting entire genes into genomes to overcome diverse disease-causing mutations – ScienceDaily

Many genetic diseases are caused by diverse mutations that spread across an entire gene, and tailoring genome editing approaches to each patient’s mutation would be impractical and expensive.

Researchers at Massachusetts General Hospital (MGH) have recently developed an improved method that improves the accuracy of inserting large segments of DNA into the genome.

This approach could be used to introduce a full natural or ‘wild-type’ replacement gene, which could serve as a universal cure for a disease regardless of the patient’s specific mutation.

The work includes optimizing a new class of technologies called CRISPR-associated transferases (CASTs), which are promising tools for inserting large, easily targeted DNA into a desired genomic location via reprogrammable guide RNA.

In their natural state, however, CASTs have characteristics that are undesirable for genome editing applications—namely, suboptimal product purity (the number of times only the intended DNA sequence is inserted into the genome) and a relatively high rate of undesirable off-target integration into sites unintended in the genome.

In their research published in Nature Biotechnologya team led by first author Connor Tu, a graduate student at MIT and MGH, and senior author Ben Kleinstefer, PhD, research assistant in the Center for Genomic Medicine at MGH and assistant professor at Harvard Medical School, addressed these shortcomings by using engineering approaches. Protein to modify the properties of CAST systems.

They found that adding a specific enzyme called a directed endonuclease to the CASTs significantly increased the purity of the product toward the intended insertion.

Further optimization of the structure of CASTs resulted in DNA insertions with high integration efficiencies at the intended genetic targets with significantly reduced insertions at unwanted off-target sites.

The researchers named the new and improved system “HELIX,” which stands for Homing Endonuclease-Assisted Large-Sequence Integrating CAST-compleX.

“We have demonstrated a generalizable approach that can be used to modify a variety of CAST regimens into safer and more effective versions with high product purity and genome-wide specificity,” says Tu.

“Combining our insights, we created HELIX systems with an integration specificity of more than 96% on target—increased from about 50% for the naturally occurring wild-type CAST system. We also determined that HELIX maintains its beneficial properties in human cells.”

Kleinstievre notes that this technology could have applications beyond the ability to restore normal healthy genes to individuals with disease-causing mutations.

“In addition, programmable DNA integration can facilitate cell engineering efforts as fixation of large gene sequences at target sites can endow cells with new capabilities while avoiding safety, efficacy, and manufacturing issues arising from traditional random integration approaches,” he says.

The study was also co-authored by Beno Orr.

This work was supported by the National Science Foundation and MGH.

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