CRISPR revolutionized gene editing. Now his toolbox is expanding

The gene editing tool that revolutionized biology becomes even more powerful.

CRISPR, as a known system, allows scientists to target and cut out a specific sequence of letters on a strand of DNA with unprecedented accuracy. This has opened up new possibilities for treating genetic diseases, helping plants adapt to global warming and preventing even mosquitoes from spreading malaria.

CRISPR is composed of two basic components. The first is a piece of RNA that locates a predetermined sequence of DNA in the genome of an organism that scientists want to modify. The second is a type of protein called enzyme that attaches to the target section of the DNA and splices it.

Cas9 is the spearhead of the enzyme because it performs a clean and crisp cut. But in recent years, scientists have begun to research – and find – alternative CRISPR systems using enzymes other than Cas9.

"Cas9 is a powerful tool, but it has limitations," said Feng Zhang, a CRISPR pioneer, a bioengineering engineer at MIT and the Broad Institute. "Each of these proteins has weaknesses and strengths, and together they help us create a much more versatile toolbox."

Some of the new Cas enzymes cut DNA in different ways, making some modifications more likely to work. Other enzymes are smaller, which allows scientists to insert them more easily into the cells.

"The CRISPR protein diversity is exceptionally large," said Benjamin Oakes, badociate researcher at the Innovative Genomics Institute, a joint project of the University of California at Berkeley and the University of California at San Francisco. "They have evolved over the millennia and nature has developed hundreds, if not thousands, that can work."

In nature, bacteria use this technology as a defense mechanism to detect and destroy attacking viruses.

The bacteria store viral DNA sequences in their own DNA, followed by a repeated letter sequence. Hence the name of the CRISPR system, which means "Regularly spaced and regularly spaced short palindromic repeats". (The first discovered CRISPR systems were actually partially palindromic, but scientists later discovered that this was not universally true.)

CRISPR-Cas9 has already been shown to be an extremely useful tool for a wide variety of genetic modifications, including the activation and deactivation of genes, their complete deactivation, the introduction of a new DNA in a genome and the removal of unwanted DNA.

But scientists wondered what other CRISPR enzymes could bring to the genetic editing table.

CRISPR-Cas12a was the first system after CRISPR-Cas9 to be used for gene editing in the laboratory. A recent study on Cas12a's cousin, Cas12b, showed that this variant could also modify the human genome, giving scientists another tool to fight against genetic diseases.

Other work has highlighted a series of additional promising CRISPR enzymes, including Cas13, Cas14 and CasY. The last candidate, CasX, was described in detail Monday in a study by Oakes and others in the journal Nature.

Comparing CRISPR systems, it's a lot like comparing fruits, Zhang said. If the Cas9 enzymes are apples, then the Cas12 enzymes could be plums – still edible and delicious, but also totally different.

And like fruits, these different systems have variations. Just as there are subspecies of plums, there is also a wide variety of Cas12 enzymes.

These differences are manifested in many ways. For example, unlike CRISPR-Cas9, which cuts both strands of DNA at the same place, CRISPR-Cas12a and CRISPR-CasX perform what's called a staggered cut, so the two strands are cut in different places.

There is evidence that a staggered break increases the chances that a cell will accept a new fragment of DNA at the splice site, said Thomas Clements, a University geneticist. Vanderbilt of Nashville. So, if the goal is to add DNA to a cell, a system using Cas12a, and perhaps a CasX day, might be a better choice than Cas9, he said. declared.

The sizes of the different CRISPR proteins also vary. In the Cas9 family, there are enzymes composed of 1,350 nucleotides (the basic structural unit of DNA) and others containing only 1,000 nucleotides. CasX and Cas12a are even smaller.

Size is an important criterion because smaller molecules are easier to enter cells than larger ones, said Eugene Koonin, who is studying evolutionary genomics at the National Institutes of Health.

"In a sense, there is a race to find the smallest effective genome publisher so you can combine it with other elements that you can pack into a viral vehicle" for an in-house delivery. A cell, he said. "Size is critical."

Scientists are exploring the planet for alternatives to Cas9.

Jillian Banfield, a geomicrobiologist at UC Berkeley, discovered that CasX and CasY were integrated into the DNA of bacteria that live in underground aquifers.

One of the earliest known forms of Cas12b was discovered in the clean room where NASA's Viking probe was badembled.

Scientists are also sampling organisms living in lakes, rivers and marine environments, as well as sites hundreds of meters below the surface of the Earth, to identify the inventive gene writers that could be harboring bacteria.

"We are exploring all the diversity of Earth's habitable environments in order to sample organisms through the tree of life," she said.

There are already thousands of known Cas9 variants and even more variants of Cas12 that still need to be studied and characterized. There are also researchers who tinker with known CRISPR systems to make them more efficient.

"There are plenty of opportunities to explore how we might be able to improve the CRISPR toolbox," Feng said. "The more options people have, the more likely we are to treat as many diseases as possible."

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