2019-05-01_Discover

MAY 2019. DISCOVER 31

Genetic Fix 101

Some genetic diseases, like hypertrophic cardiomyopathy, are passed on from one generation to another by a single defective gene.

A father with one bad copy of the gene would have a 50 percent chance of passing it on to his children. Mitalipov’s group tested how

CRISPR-Cas9 might allow them to repair a defective gene from a disease-carrying sperm right as it fertilizes a healthy egg.

Eggs and sperm carry half of a complete genome

— one copy of each gene. Here, the sperm carries

a disease-causing gene.

The egg is injected with CRISPR gene repair

tools: the gene-cutter Cas9, a guide RNA and

DNA templates for the normal gene.

Cas9 is an enzyme that will cut DNA in a precise

location, according to the search function built

into the CRISPR guide.

During fertilization, the half-genomes from

the egg and sperm will combine to form one

complete human genome.

At some point in this process, Cas9 finds the

defective gene that matches the CRISPR guide

and causes a double-strand break.

Detecting the DNA break, the cell attempts repair.

But instead of using the repair template provided

by researchers, it uses the maternal DNA.

But only recently has there been a clear way to do it.

The earliest work on what would become CRISPR (short

for clustered regularly interspaced short palindromic repeats)

happened some 30 years ago, but it took researchers nearly

all that time to figure out the full CRISPR-Cas9 system and

to begin harnessing it for gene editing. The system of DNA

sequences occurs naturally in bacteria, helping them fight

off attacking viruses. Bacteria incorporate a small chunk of

DNA when they encounter a specific virus, a little souvenir

to remember their viral attacker in the future. The bacteria’s

defense system includes a seek-and-destroy function that

uses the viral DNA as a search image. Part of the mechanism

includes production of the protein Cas9, which snips the DNA

that matches the template. For a virus trying to infiltrate a

bacterial cell, this means game over.

Today, biologists have learned to reprogram CRISPR-Cas9 to

cut any type of DNA in a cell — not just viral — in a location of

their choosing by giving it a new target to seek out. They’ve also

discovered that after the DNA is cut by Cas9, cells will try to repair

the break in the DNA. That repair system can then be manipulated

into using a template provided by scientists, effectively cutting out

one gene and replacing it with another.

Mitalipov and like-minded colleagues believe the promise of

CRISPR is that they will be able to use it to replace a defective

gene with a functioning one. To test this, the OHSU team’s

experiments, published in the journal Nature, were straightfor-

ward. Using sperm from a man carrying the defective MYBPC3

gene and eggs from a healthy woman, they would see if they

could use CRISPR-Cas9 to repair the disease-causing gene.

They injected each egg with a sperm carrying the mutation

and a CRISPR-Cas9 package. In this case, the package included

the DNA search image that would help Cas9 find the defective

gene. They also included a sequence of DNA that matched

the normal version of the gene, which the cell uses as a repair

template to mend the cut in its DNA. They added a little calling

card to this repair template — swapping out two nucleotide

bases that would change the sequence, but not the function,

of the normal gene. With this, they could know whether the

cell used their template.

Their experiments worked, but not in the way they expected.

Cas9 did locate and cut the disease-causing gene the embryo

had inherited from its father. But instead of using the template

the researchers provided, the embryo used the normal gene

from the mother as a template, resulting in two normal genes.

However, some scientists remain skeptical the experiments

worked as well as Mitalipov’s group claimed because of the

difficulties of confirming that the gene editing went as planned.

Their biggest holdup? It’s possible that instead of two normal

JAY

SM

ITH

CRISPR-Cas9 System

repair template

Cas9 binds

to

Cas9

guide

RNA

target sequence

of mutated gene

maternal

DNA

paternal

DNA double strand

break

unused repair

template

paternal

DNA

maternal

DNA

healthy

gene