The study reveals limitations in evaluating gene editing technology in human embryos

A commonly used scientific method to analyze the tiny amount of DNA in early human embryos does not accurately reflect gene changes, according to new research led by scientists at Oregon Health & Science University.

The study, published today in the journal Nature Communication, involved the genome sequencing of early human embryos that had been genome edited using the gene editing tool CRISPR. The work questions the accuracy of a DNA reading procedure that relies on amplifying a small amount of DNA for genetic testing purposes.

In addition, the study shows that gene editing to correct disease-causing mutations in early human embryos can lead to unintended and potentially harmful changes in the genome.

Together, the results raise a new scientific basis to warn any scientist who might be able to use genetic embryos to establish a pregnancy. Although gene editing technologies hold promise for preventing and treating debilitating inherited diseases, the new study shows limits that must be overcome before gene editing can be considered safe or effective for establishing pregnancy.

“It tells you how little we know about editing the genome, and especially how cells respond to CRISPR-induced DNA damage,” said senior author Shoukhrat Mitalipov, Ph.D., director of the OHSU Center for Embryonic Cell and Gene Therapy; and, professor of obstetrics and gynecology, molecular and cellular biological sciences, OHSU School of Medicine, OHSU Oregon National Primate Research Center. “Gene repair has great potential, but these new results show that we have a lot of work to do.”

The findings come during the Third International Summit on Human Genome Editing in London. On the eve of the last international summit, held in Hong Kong in November 2018, a Chinese scientist revealed the birth of the world’s first children as a result of gene-edited embryos through an experiment that caused global criticism.

Misdiagnosed embryos

Before an edited embryo can be transferred to establish a pregnancy, it is important to ensure that the procedure works as intended.

Because early human embryos contain only a few cells, it is not possible to collect enough genetic material to analyze them effectively. Instead, scientists interpret data from a small sample of DNA taken from a single or even a single cell, which must then be multiplied millions of times during a process called whole genome amplification.

The same process—called preimplantation genetic testing, or PGT—is often used to screen human embryos for various genetic conditions in patients undergoing in vitro fertilization.

Whole genome amplification has limitations that reduce the accuracy of genetic testing, said co-senior author Paula Amato, MD, professor of obstetrics and gynecology at the OHSU School of Medicine.

“The concern is that we could be misdiagnosing embryos,” Amato said.

Amato, who uses in vitro fertilization to treat patients struggling with infertility as well as prevent the transmission of hereditary diseases, said PGT using more advanced technology is still clinically useful for abnormalities detection of chromosomal and genetic disorders caused by a single gene mutation transmitted from a parent. his child.

The study highlights the challenges of establishing the safety of gene editing techniques.

“We may not be able to reliably predict that this embryo will result in a healthy child,” Mitalipov said. “That’s a big problem.”

To overcome these issues, OHSU researchers, along with collaborators with research institutions in South Korea and China, established embryonic stem cell lines from gene-edited embryos. Embryonic stem cells grow indefinitely and provide an abundance of DNA material that does not require whole genome amplification for analysis.

Researchers say the discovery highlights the erratic nature of whole-genome amplification and the need to verify changes in embryos by establishing embryonic stem cell lines.

The study verifies gene repair

Using embryonic stem cells, the new study verifies the gene repair process developed by Mitalipov’s lab; The results were published in the journal nature in 2017 and verified in 2018.

In that study, the scientists cut a specific target sequence of a mutant gene known to be carried by a sperm donor.

​​​​Researchers found that human embryos repair these breaks, using the normal copy of the gene from the other parent as a template. Mitalipov and co-authors confirmed that this process, known as gene conversion, occurs regularly in human embryos soon after a double-strand break in their DNA. Such a repair, if used to establish a pregnancy through in vitro fertilization and embryo transfer, could theoretically prevent the transmission of a known family disease to the child, as well as all future generations of the family .

In the study published in 2017, the OHSU researchers focused on a gene known to cause fatal heart disease.

In this new publication, the researchers focused on other isolated mutations using donated sperm and eggs, including one known to cause hypertrophic cardiomyopathy, a condition in which the heart muscle becomes too thick , and another condition associated with high cholesterol. In each case an enzyme called Cas9, used in conjunction with CRISPR, triggered a double-strand break in DNA at the exact location of the mutation.

Creating problems

In addition to replicating and confirming the gene repair mechanism reported in 2017, the new study examines what happens in the genome beyond the specific location where the mutant gene is repaired. And that’s where there can be a problem.

“In this paper we asked, ‘how extensive is that gene conversion repair mechanism?'” Amato said. “It turns out that it can be very long.”

Extensive copying of the genome, from one parent to the other, creates a situation known as loss of heterozygosity.

Everyone shares two versions, or alleles, of every gene on the human genome—one that each parent contributes. Most of the time, the alleles represent 99.9% of any one’s DNA sequence shared with the rest of humanity. In some cases, however, one parent will carry a recessive disease-causing mutation that is usually canceled out by the other parent’s dominant healthy version of the same gene.

These polymorphisms in the genetic code can be critical. For example, a gene may encode a protein that protects against specific types of cancer.

“If you have one abnormal copy of a recessive mutation, there may be no risk,” Amato said. “But if you have a loss of heterozygosity that results in two mutant copies of the same tumor-promoting gene, you are now at a significantly increased risk of cancer.”

The more genetic code that is copied, the greater the risk of dangerous genetic changes. In the new study, the scientists measured areas of gene conversion from a relatively small segment to as large as 18,600 base pairs of DNA.

In fact, fixing one known mutation may cause more problems than it solves.

“If you are cutting in the middle of a chromosome, there could be 2,000 genes,” said Mitalipov. “You’re fixing one tiny spot, but all these thousands of genes upstream and downstream could be affected.”

The result suggests that much more research is needed to understand the mechanism at work in gene editing before it can be used clinically to establish pregnancy.