Mini CRISPR system promises easier cell engineering, gene therapy

14 mins read

Scientists have developed a more compact CRISPR system that can be more easily delivered to cells.

The technology, described Friday in the journal Molecular Cell, promises to make cell-engineering more effective and efficient.

The world’s most popular and powerful gene-editing tool, CRISPR-Cas9, has enabled numerous scientific discoveries. It works by excising specific DNA sequences using a gene-cutting protein, Cas9, sourced from bacterial immune systems.

Because CRISPR-Cas9 and its derivatives are sometimes too large and cumbersome to be used in certain cellular environs, limiting their adaptability, researchers at Stanford University developed a smaller CRISPR-Cas system called CasMINI.

“This is a critical step forward for CRISPR genome-engineering applications,” senior study author Stanley Qi said in a press release.

“The work presents the smallest CRISPR to date, according to our knowledge, as a genome-editing technology. If people sometimes think of Cas9 as molecular scissors, here we created a Swiss knife containing multiple functions. It is not a big one, but a miniature one that is highly portable for easy use,” Qi said.

Scientists suggest their new gene-editing system will allow researchers to develop gene therapies that couldn’t be delivered using previous CRISPR systems.

To shrink their system, scientists turned to a gene-cutting protein called Cas12f, or Cas14. The protein is composed of between 400 and 700 amino acids, making it less than half the size of more traditional gene-editing proteins like Cas9 or Cas12a.

Until recently, scientists weren’t sure whether Cas14 could operate inside mammalian cells.

“Recent years have identified thousands of CRISPRs, which are known as bacteria’s immunity defense system,” Qi said. “More than 99.9% of discovered CRISPRs, however, cannot work in human cells, limiting their use as genome-editing technologies.”

After confirming that Cas12f is inactive inside mammalian cells, researchers subjected the protein to several rounds of engineering, until they derived a variant capable of gene-regulation and gene-editing.

“Here we turn a non-working CRISPR in mammalian cells, via rational RNA engineering and protein engineering, into a highly efficient working one,” Qi said.

“There were previous efforts from others to improve the performance of working CRISPRs. But our work is the first to make a non-working one working. This highlights the power of bioengineering to achieve something evolution has not yet done,” Qi said.

The new Cas12f variant, dubbed CasMINI, features just 529 amino acids. Its compact size allows it to be easily smuggled into a variety of target cells and can be carried into cells using an adeno-associated virus or lipid nanoparticles.

Scientists are currently testing how different delivery systems impact the protein’s gene-regulation and gene-editing abilities once inside the target cell.

“The availability of a miniature CasMINI enables new applications, ranging from in vitro applications such as engineering better tumor-killing lymphocytes or reprogramming stem cells to in vivo gene therapy to treat genetic diseases in the eye, muscle, or liver,” Qi said.

“It is on our wish list that it will become a therapy to treat genetic diseases, to cure cancer, and to reverse organ degeneration,” Qi said.

 

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