Building on the CRISPR gene-editing system, MIT researchers have designed a new tool that
can snip out faulty 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 as long as 36,000
DNA base pairs to several types of human cells, as well as to liver cells in mice.
The new technique, known as PASTE, could hold promise for treating diseases that are caused
by defective genes with a large number of mutations, such as cystic fibrosis.
The new tool combines the precise targeting of CRISPR-Cas9, a set of molecules originally
derived from bacterial defense systems, with enzymes called integrases, which viruses use
to insert their own genetic material into a bacterial genome.
Just like CRISPR, these integrases come from the ongoing battle between bacteria and the
viruses that infect them.
The CRISPR-Cas9 gene editing system consists of a DNA-cutting enzyme called Cas9 and a
short RNA strand that guides the enzyme to a specific area of the genome, directing Cas9
where to make its cut.
When Cas9 and the guide RNA targeting a disease gene are delivered into cells, a specific
cut is made in the genome, and the cells’ DNA repair processes glue the cut back together,
often deleting a small portion of the genome.
If a DNA template is also delivered, the cells can incorporate a corrected copy into their
genomes during the repair process.
However, this process requires cells to make double-stranded breaks in their DNA, which
can cause chromosomal deletions or rearrangements that are harmful to cells.
Another limitation is that it only works in cells that are dividing, as nondividing cells
don’t have active DNA repair processes.
The MIT team wanted to develop a tool that could cut out a defective gene and replace
it with a new one without inducing any double-stranded DNA breaks.
To achieve this goal, they turned to a family of enzymes called integrases, which viruses
called bacteriophages use to insert themselves into bacterial genomes.
For this study, the researchers focused on serine integrases, which can insert huge chunks
of DNA, as large as 50,000 base pairs.
These enzymes target specific genome sequences known as attachment sites, which function
as “landing pads.”
When they find the correct landing pad in the host genome, they bind to it and integrate
their DNA payload.
In past work, scientists have found it challenging to develop these enzymes for human therapy
because the landing pads are very specific, and it’s difficult to reprogram integrases
to target other sites.
The MIT team realized that combining these enzymes with a CRISPR-Cas9 system that inserts
the correct landing site would enable easy reprogramming of the powerful insertion system.
The new tool, PASTE (Programmable Addition via Site-specific Targeting Elements), includes
a Cas9 enzyme that cuts at a specific genomic site, guided by a strand of RNA that binds
to that site.
This allows them to target any site in the genome for insertion of the landing site,
which contains 46 DNA base pairs.
This insertion can be done without introducing any double-stranded breaks by adding one DNA
strand first via a fused reverse transcriptase, then its complementary strand.
Once the landing site is incorporated, the integrase can come along and insert its much
larger DNA payload into the genome at that site.
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 could be
therapeutically useful, and were able to insert them into nine different locations in the
genome.
In these cells, the researchers were able to insert genes with a success rate ranging
from 5 to 60 percent.
This approach also yielded very few unwanted “indels” (insertions or deletions) at
the sites of gene integration.
The researcher says that they see very few indels, and because they’re not making double-stranded
breaks, we don’t have to worry about chromosomal rearrangements or large-scale chromosome arm
deletions.
The researchers also demonstrated that they could insert genes in “humanized” livers
in mice.
Livers in these mice consist of about 70 percent human hepatocytes, and PASTE successfully
integrated new genes into about 2.5 percent of these cells.
The DNA sequences that the researchers inserted in this study were up to 36,000 base pairs
long, but they believe even longer sequences could also be used.
A human gene can range from a few hundred to more than 2 million base pairs, although
for therapeutic purposes only the coding sequence of the protein needs to be used, drastically
reducing the size of the DNA segment that needs to be inserted into the genome.
The researchers are now further exploring the possibility of using this tool as a possible
way to replace the defective cystic fibrosis gene.
This technique could also be useful for treating blood diseases caused by faulty genes, such
as hemophilia and G6PD deficiency, or Huntington’s disease, a neurological disorder caused by
a defective gene that has too many gene repeats.
The researchers have also made their genetic constructs available online for other scientists
to use.