Nature sub-issue: MIT's newly developed CRISPR-based "paste" tool

Building on the CRISPR gene-editing system, MIT researchers have devised a new tool that can cut out defective genes and replace them
with new ones in a safer and more efficient way.
Using this system, the researchers showed that they could deliver genes for up to 36,000 DNA base pairs to several types of human cells, as well as liver cells
from mice.
The new technique, called PASTE, is expected to be used to treat diseases caused by a large number of mutated defective genes, such as cystic fibrosis
.

Omar Abudayyeh, a McGovern Fellow at MIT's McGovern Brain Institute, said: "This is a new genetic approach that could potentially target these difficult-to-treat diseases
.
We want to work in the direction that gene therapy should do in the first place, which is to replace genes, not just correct individual mutations
.
”

The new tool combines precise targeting of CRISPR-Cas9, a group of molecules originally derived from the bacterial defense system, and an enzyme called integrase, which viruses use to insert their own genetic material into the bacterial genome
.

"Just like CRISPR, these integrases come from the ongoing battle between bacteria and the virus that infects them," said
McGovern researcher Jonathan Gootenberg.
"This speaks to how we continue to discover a large number of interesting and useful new tools
from these natural systems.
"

Gootenberg and Abudayyeh, senior authors of the new study, published today in
Nature Biotechnology.

DNA insertion

The CRISPR-Cas9 gene-editing system consists of a DNA-cutting enzyme called Cas9 and a short RNA strand that guides the enzyme to specific regions of the genome, directing where Cas9 is cut
.
When Cas9 and guide RNA targeting disease genes are delivered into the cell, there is a specific cut in the genome that the cell's DNA repair process binds together, often deleting a small part
of the genome.

If the DNA template is also delivered, cells can incorporate a modified copy into
their genome during repair.
However, this process requires cells to make double-strand breaks in DNA, which can lead to chromosomal deletions or rearrangements
that are harmful to the cell.
Another limitation is that it only works in dividing cells, because undivided cells do not have an active DNA repair process
.

The MIT team wanted to develop a tool that could cut out a defective gene without causing any double-stranded DNA breaks and replace it
with a new one.
To achieve this, they turned to a family of enzymes called integrase, viruses called bacteriophages used to insert into bacterial genomes
.

In this study, the researchers focused on serine integrase, which can insert huge blocks of DNA, as large as 50,000 base pairs
.
These enzymes target specific genome sequences called attachment sites, which act as
"landing pads.
" When they find the right landing site in the host genome, they bind to it, integrating their DNA load
.

In past work, scientists have found it challenging to develop these enzymes for human treatment because landing pads are so specific that it is difficult to reprogram integrase to locate other sites
.
The MIT team realized that combining these enzymes with a CRISPR-Cas9 system inserted into the correct landing site would make the powerful insertion system easy to reprogram
.

The new tool, PASTE (programmable to add a targeted element through site-specific), includes a Cas9 enzyme that cleaves specific genomic loci under the guidance of RNA strands bound to that site
.
This allows them to target any location in the genome to insert landing sites, which contain 46 DNA base pairs
.
This insertion can be done without introducing any double-strand breaks, first by adding a DNA strand by a fused reverse transcriptase, followed by its complementary strand
.

Once the landing site is integrated, the integrase emerges and inserts its larger DNA payload into the genome of that site
.

"We see this as a big step toward realizing the dream of programmable DNA insertion," Gootenberg said
.
"This technology can be easily customized
according to the website and goods we want to integrate.
"

Gene replacement

In this study, the researchers showed that they could use PASTE to insert genes into several types of human cells, including liver cells, T cells, and lymphoblasts (immature white blood cells).

They tested the delivery system with 13 different payload genes, including some that may have therapeutic value, and were able to insert them into 9 different locations
in the genome.

In these cells, the researchers were able to insert genes
with a success rate of 5 to 60 percent.
This approach also produces few unwanted "insertions" (insertions or deletions)
at sites of gene integration.

"We see very few inner links because we don't have double-strand breaks, and you don't have to worry about chromosomal rearrangements or massive chromosomal arm deletions
," Abudayyeh said.

The researchers also demonstrated that they could insert genes
into the livers of "humanized" mice.
The livers of these mice are made up of about 70 percent of human liver cells, and PASTE successfully integrated the new gene into 2.
5 percent
of these cells.

The DNA sequences the researchers inserted in this study were up to 36,000 base pairs, but they believe even longer sequences
can be used.
The number of base pairs of human genes ranges from a few hundred to more than 2 million, although for therapeutic purposes only the coding sequence of the protein needs to be used, which greatly reduces the size of
the DNA fragment that needs to be inserted into the genome.

The researchers are now further exploring the possibility
of using this tool to replace the defective cystic fibrosis gene.
The technique could also be used to treat blood disorders caused by defective genes, such as hemophilia and G6PD deficiency, or Huntington's disease, a neurological disorder
caused by too many gene duplications in the defective gene.

The researchers also put their genetic makeup online for other scientists to use
.

"The wonderful thing about engineering these molecular techniques is that people can build on them and develop and apply them in ways that we may not have thought of or considered," Gootenberg said
.
"It's amazing
to be part of this emerging community.
"

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases