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July 31, 2018

DNA Crosslink Repair Central to CRISPR Insert Success

  • In CRISPR-Cas9 gene editing, the balance between two DNA repair pathways gets all the attention. Everybody wants to know, when Cas9 makes a cut, will DNA be repaired by non-homologous end joining (NHEJ), a quick-and-dirty procedure, or by homology-directed repair (HDR), a relatively painstaking procedure guided by template DNA. Well, it turns out that a third pathway can tilt the balance toward NHEJ or HDR, influencing the success rates of gene editing applications, including those with therapeutic potential.

    Although the third pathway has been overlooked in the gene editing context, it has long been recognized for its role in Fanconi anemia. This pathway, which involves 21 separate proteins, is called the Fanconi anemia pathway because, if any of the genes for these proteins is damaged, people develop Fanconi anemia, a rare but serious hereditary disease in which the bone marrow cannot make enough new blood cells. It is associated with birth defects and an elevated risk of cancer, including a 10% chance of developing leukemia in childhood. Few patients live beyond 30 years of age.

    The pathway has been known and studied for decades, but it was largely understood to repair one specific kind of DNA damage: DNA interstrand crosslinks, where a nucleotide on one strand of DNA bonds tightly with a nucleotide on the adjacent strand, interfering with DNA replication and often killing the cell. Researchers in the 1980s had reported a connection between homology-directed repair and the Fanconi anemia pathway, but it had been ignored or misunderstood.

    The Fanconi anemia pathway’s overlooked role in CRISPR-Cas9 applications came to light in a recent study conducted by scientists based at the University of California, Berkeley. These scientists, hoping to improve our understanding of NHEJ and HDR—and why one might predominate over the other—decided to systematically explore the functionality of genes known or suspected to be involved in DNA repair. Led by Jacob Corn, Ph.D., assistant adjunct professor of biochemistry, biophysics and structural biology, University of California, Berkeley, these scientists employed a technique called CRISPR interference (CRISPRi) to knock out, one at a time, more than 2000 genes of interest.

    The results of this work appeared online July 27 in the journal Nature Genetics, in an article titled, “CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway.” According to this article, one of the 21 proteins in the Fanconi anemia pathway, FANCD2, always homes in on the site of the double-strand break created by CRISPR-Cas9, indicating it plays an important role in regulating the insertion of new DNA into the genome at the cut site. FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.

    “The Fanconi anemia pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward single-strand template repair,” wrote the article’s authors. “Furthermore, FANCD2 protein localizes to Cas9-induced double-strand breaks, indicating a direct role in regulating genome editing.”

    The importance of the Fanconi anemia pathway in repairing CRISPR breaks throws into doubt some planned CRISPR treatments for the disease itself. Without an active Fanconi anemia pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut.

    In fact, the level of activity of the Fanconi anemia pathway may affect how efficiently CRISPR can insert DNA in a specific cell. The researchers concluded that, while end-joining is the default repair mechanism after a double-strand break, the Fanconi anemia pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.

    "The enthusiasm for using CRISPR-Cas9 for medical or synthetic biology applications is great, but no one really knows what happens after you put it into cells," said Chris D. Richardson, Ph.D., the first author of the current paper. "It goes and creates these breaks and you count on the cells to fix them. But people don't really understand how that process works."

    "Gene editing is super-powerful, with a lot of promise, but, so far, a lot of trial and error. The way it works in human cells has been a black box with a lot of assumptions," added Dr. Corn. "We are finally starting to get a picture of what's going on."

    The findings not only shed light on the DNA repair mechanisms in human cells, they also suggest how anticancer therapies could target DNA repair in cancer cells. Because other factors now appear to be involved in the repair of double-strand breaks, this research expands the list of proteins that could be misregulated to disrupt DNA repair in cancer cells and make them more susceptible to death.

    "The whole Fanconi anemia pathway affects the balance between end-joining and homology-directed repair; it acts like a traffic cop," added Dr. Corn. "So, a patient's genotype will affect how you do gene editing."

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