Study reveals limitations in evaluating gene-editing technology in human embryos — ScienceDaily

A commonly used scientific method for analyzing a tiny amount of DNA in early human embryos fails to accurately reflect gene modifications, according to new research led by scientists at Oregon Health & Science University.

The study, published today in the journal Nature communications, involved sequencing the genomes of early human embryos that had been genome edited using the CRISPR gene editing tool. The work calls into question the accuracy of a DNA reading process that relies on amplifying a small amount of DNA for genetic screening purposes.

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

Together, the findings create a new scientific basis for caution for any scientist who may be ready to use genetically engineered embryos to create pregnancies. Although gene-editing technologies hold promise for preventing and treating debilitating inherited diseases, the new study reveals limitations that must be overcome before gene editing to detect a pregnancy can be considered safe or effective.

“It tells you how little we know about genome editing and particularly 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 life sciences, OHSU School of Medicine, OHSU Oregon National Primate Research Center. “Gene repair has great potential, but these new results show we have a lot of work to do.”

The findings emerged during the Third International Summit on Human Genome Editing in London. On the eve of the latest international summit, held in Hong Kong in November 2018, a Chinese scientist revealed the birth of the world’s first babies from genetically engineered embryos through an experiment that drew global condemnation.

Misdiagnosis of embryos

Before a processed embryo can be transferred to establish a pregnancy, it is important to make sure that the procedure worked as it should.

Because early human embryos consist of 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 few or even a single cell, which must then be amplified millions of times during a process known as whole-genome amplification.

The same procedure — known as preimplantation genetic testing, or PGT — is often used to screen human embryos for various genetic conditions in IVF patients.

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

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

Amato, who uses IVF to treat patients struggling with infertility as well as prevent the transmission of inherited diseases, said PGT using more advanced technology is still clinically useful for detecting chromosomal abnormalities and genetic disorders caused by from a single gene mutation passed down from the parent. to the kid.

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 lead to a healthy baby,” Mitalipov said. “That’s a big problem.”

To overcome these issues, OHSU researchers, along with collaborators with research institutions in South Korea and China, created lines of embryonic stem cells from gene-edited embryos. Embryonic stem cells grow indefinitely and provide abundant DNA material that does not require whole-genome amplification to be analyzed.

The researchers say the discovery highlights the error-prone nature of whole-genome amplification and the need to verify modifications in embryos by creating embryonic stem cells.

Study validates gene repair

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

In this study, the scientists cut out a specific target sequence in a mutated gene known to be carried by a sperm donor.

The 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 early human embryos after a double-strand break in their DNA. Such a fix, if used to create a pregnancy through IVF and embryo transfer, could theoretically prevent the transmission of a known family disease to the child, as well as to all future generations of the family.

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

In this new paper, the researchers targeted other distinct mutations using sperm and eggs, including a mutation known to cause hypertrophic cardiomyopathy, a condition in which the heart muscle becomes abnormally thick, and another associated with high cholesterol. In each case, an enzyme known as Cas9, used in conjunction with CRISPR, caused a double-stranded break in the DNA at the exact location of the mutation.

Creating problems

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

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

The extensive copying of the genome, from one parent to the other, creates a scenario known as loss of heterozygosity.

Every human being shares two versions, or alleles, of every gene in the human genome — one from each parent. Most of the time, the alleles are identical, since 99.9% of any individual’s DNA sequence is 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 extremely important. For example, a gene may code for a protein that protects against certain types of cancer.

“If you have an abnormal copy of a recessive mutation, that may not be a risk,” Amato said. “But if you have loss of heterozygosity that results in two mutated copies of the same tumor suppressor gene, you are now at a significantly increased risk for cancer.”

The more genetic code is copied, the greater the risk of dangerous genetic changes. In the new study, the scientists measured gene conversion pathways ranging from a relatively small stretch of up to 18,600 base pairs of DNA.

In fact, fixing a known mutation can create more problems than it solves.

“If you cut in the middle of a chromosome, there could be 2,000 genes there,” Mitalipov said. “You fix a tiny spot, but all those thousands of genes upstream and downstream can be affected.”

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

Studies conducted at the OHSU Center for Embryonic Cell and Gene Therapy were supported by OHSU institutional funds and a grant from the Burroughs Wellcome Fund.

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