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New research suggests the Fanconi anemia (FA) pathway plays a key role in repairing double-strand breaks (DSBs) created by CRISPR-Cas9 genome editing.
Researchers said they found that Cas9-induced single-strand template repair requires the FA pathway, and the protein FANCD2 localizes to Cas9-induced DSBs.
The team said this research provides insight into why CRISPR-Cas9 does not produce equal success in all cells.
Furthermore, the work might help researchers boost the efficiency with which cells insert new DNA into the genome and generally tweak CRISPR-Cas9 editing to get the desired outcome.
“If you want to treat sickle cell anemia, your chances of success are inextricably tied to the efficiency with which you can replace the mutated sickle cell gene with the correct one,” said study author Chris Richardson, PhD, formerly of the University of California, Berkeley, but now at Spotlight Therapeutics in Hayward, California.
“If you harvest a million cells from a patient and you have a 10% insertion rate, that is not as good as if you have 30% to 40%. Being able to manipulate those cells to increase the frequency of this process, called homology-directed repair, is exciting.”
Dr Richardson and his colleagues described this work in Nature Genetics.
CRISPR relies on DNA repair
The researchers noted that CRISPR-Cas9 creates targeted DSBs, and it’s up to the cell to repair the DNA.
This can happen in 2 ways. Enzymes can stitch the dangling ends back together, which often results in one or more bases being added or deleted, disrupting the function of the gene.
Alternatively, other enzymes can patch the break with a single strand of DNA that matches the DNA sequence upstream and downstream of the cut. A complementary DNA strand is created to complete the double-strand repair.
The former method, called non-homologous end-joining, appears to be the most common outcome after CRISPR cutting.
The latter method, homology-directed repair, happens more frequently in some cells than others and requires the presence of DNA that can be used to patch the break. Researchers often supply a single-stranded piece of DNA and hope the cell uses it to replace the faulty sequence with the new one.
Both processes are a bit mysterious, however, and no one knows why some cells readily patch in DNA while others do so infrequently.
“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,” Dr Richardson said. “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.”
To find out which DNA repair enzymes are critical to homology-directed repair after CRISPR cutting, Dr Richardson and his colleagues used a technique called CRISPR interference. They knocked out, one at a time, more than 2000 genes known or suspected to be involved in DNA repair.
The researchers were surprised to find that many of the genes that proved important—homology-directed repair dropped dramatically when they were silenced—were involved in the FA pathway.
FA pathway
The FA pathway was largely understood to repair 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.
“Based on our work, we believe that the Fanconi anemia pathway plays a major role in fixing other types of lesions as well, but is best understood as the pathway that repairs double-strand breaks,” Dr Richardson said. “After Cas9 editing, the Fanconi anemia pathway is required if you want to insert new DNA.”
The importance of the FA pathway in repairing DSBs casts doubt on some planned CRISPR treatments for Fanconi anemia itself.
Without an active FA pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut. In fact, the activity level of the FA 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 DSB, the FA pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.
Dr Richardson and his colleagues also found that 1 of the 21 proteins in the FA pathway, FANCD2, always homes in on the site of the DSB created by CRISPR-Cas9. This suggests FANCD2 plays an important role in regulating the insertion of new DNA at the cut site.
The researchers therefore believe FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.
“Also, since FANCD2 localizes to the site of Cas9 breaks, you can use FANCD2 to map where Cas9 is cutting in any cell type,” Dr Richardson said. “If you edit a population of cells and you want to know where the on- and off-target cuts are, you can just map where FANCD2 was found in the genome and you can find the cuts.”
New research suggests the Fanconi anemia (FA) pathway plays a key role in repairing double-strand breaks (DSBs) created by CRISPR-Cas9 genome editing.
Researchers said they found that Cas9-induced single-strand template repair requires the FA pathway, and the protein FANCD2 localizes to Cas9-induced DSBs.
The team said this research provides insight into why CRISPR-Cas9 does not produce equal success in all cells.
Furthermore, the work might help researchers boost the efficiency with which cells insert new DNA into the genome and generally tweak CRISPR-Cas9 editing to get the desired outcome.
“If you want to treat sickle cell anemia, your chances of success are inextricably tied to the efficiency with which you can replace the mutated sickle cell gene with the correct one,” said study author Chris Richardson, PhD, formerly of the University of California, Berkeley, but now at Spotlight Therapeutics in Hayward, California.
“If you harvest a million cells from a patient and you have a 10% insertion rate, that is not as good as if you have 30% to 40%. Being able to manipulate those cells to increase the frequency of this process, called homology-directed repair, is exciting.”
Dr Richardson and his colleagues described this work in Nature Genetics.
CRISPR relies on DNA repair
The researchers noted that CRISPR-Cas9 creates targeted DSBs, and it’s up to the cell to repair the DNA.
This can happen in 2 ways. Enzymes can stitch the dangling ends back together, which often results in one or more bases being added or deleted, disrupting the function of the gene.
Alternatively, other enzymes can patch the break with a single strand of DNA that matches the DNA sequence upstream and downstream of the cut. A complementary DNA strand is created to complete the double-strand repair.
The former method, called non-homologous end-joining, appears to be the most common outcome after CRISPR cutting.
The latter method, homology-directed repair, happens more frequently in some cells than others and requires the presence of DNA that can be used to patch the break. Researchers often supply a single-stranded piece of DNA and hope the cell uses it to replace the faulty sequence with the new one.
Both processes are a bit mysterious, however, and no one knows why some cells readily patch in DNA while others do so infrequently.
“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,” Dr Richardson said. “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.”
To find out which DNA repair enzymes are critical to homology-directed repair after CRISPR cutting, Dr Richardson and his colleagues used a technique called CRISPR interference. They knocked out, one at a time, more than 2000 genes known or suspected to be involved in DNA repair.
The researchers were surprised to find that many of the genes that proved important—homology-directed repair dropped dramatically when they were silenced—were involved in the FA pathway.
FA pathway
The FA pathway was largely understood to repair 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.
“Based on our work, we believe that the Fanconi anemia pathway plays a major role in fixing other types of lesions as well, but is best understood as the pathway that repairs double-strand breaks,” Dr Richardson said. “After Cas9 editing, the Fanconi anemia pathway is required if you want to insert new DNA.”
The importance of the FA pathway in repairing DSBs casts doubt on some planned CRISPR treatments for Fanconi anemia itself.
Without an active FA pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut. In fact, the activity level of the FA 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 DSB, the FA pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.
Dr Richardson and his colleagues also found that 1 of the 21 proteins in the FA pathway, FANCD2, always homes in on the site of the DSB created by CRISPR-Cas9. This suggests FANCD2 plays an important role in regulating the insertion of new DNA at the cut site.
The researchers therefore believe FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.
“Also, since FANCD2 localizes to the site of Cas9 breaks, you can use FANCD2 to map where Cas9 is cutting in any cell type,” Dr Richardson said. “If you edit a population of cells and you want to know where the on- and off-target cuts are, you can just map where FANCD2 was found in the genome and you can find the cuts.”
New research suggests the Fanconi anemia (FA) pathway plays a key role in repairing double-strand breaks (DSBs) created by CRISPR-Cas9 genome editing.
Researchers said they found that Cas9-induced single-strand template repair requires the FA pathway, and the protein FANCD2 localizes to Cas9-induced DSBs.
The team said this research provides insight into why CRISPR-Cas9 does not produce equal success in all cells.
Furthermore, the work might help researchers boost the efficiency with which cells insert new DNA into the genome and generally tweak CRISPR-Cas9 editing to get the desired outcome.
“If you want to treat sickle cell anemia, your chances of success are inextricably tied to the efficiency with which you can replace the mutated sickle cell gene with the correct one,” said study author Chris Richardson, PhD, formerly of the University of California, Berkeley, but now at Spotlight Therapeutics in Hayward, California.
“If you harvest a million cells from a patient and you have a 10% insertion rate, that is not as good as if you have 30% to 40%. Being able to manipulate those cells to increase the frequency of this process, called homology-directed repair, is exciting.”
Dr Richardson and his colleagues described this work in Nature Genetics.
CRISPR relies on DNA repair
The researchers noted that CRISPR-Cas9 creates targeted DSBs, and it’s up to the cell to repair the DNA.
This can happen in 2 ways. Enzymes can stitch the dangling ends back together, which often results in one or more bases being added or deleted, disrupting the function of the gene.
Alternatively, other enzymes can patch the break with a single strand of DNA that matches the DNA sequence upstream and downstream of the cut. A complementary DNA strand is created to complete the double-strand repair.
The former method, called non-homologous end-joining, appears to be the most common outcome after CRISPR cutting.
The latter method, homology-directed repair, happens more frequently in some cells than others and requires the presence of DNA that can be used to patch the break. Researchers often supply a single-stranded piece of DNA and hope the cell uses it to replace the faulty sequence with the new one.
Both processes are a bit mysterious, however, and no one knows why some cells readily patch in DNA while others do so infrequently.
“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,” Dr Richardson said. “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.”
To find out which DNA repair enzymes are critical to homology-directed repair after CRISPR cutting, Dr Richardson and his colleagues used a technique called CRISPR interference. They knocked out, one at a time, more than 2000 genes known or suspected to be involved in DNA repair.
The researchers were surprised to find that many of the genes that proved important—homology-directed repair dropped dramatically when they were silenced—were involved in the FA pathway.
FA pathway
The FA pathway was largely understood to repair 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.
“Based on our work, we believe that the Fanconi anemia pathway plays a major role in fixing other types of lesions as well, but is best understood as the pathway that repairs double-strand breaks,” Dr Richardson said. “After Cas9 editing, the Fanconi anemia pathway is required if you want to insert new DNA.”
The importance of the FA pathway in repairing DSBs casts doubt on some planned CRISPR treatments for Fanconi anemia itself.
Without an active FA pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut. In fact, the activity level of the FA 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 DSB, the FA pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.
Dr Richardson and his colleagues also found that 1 of the 21 proteins in the FA pathway, FANCD2, always homes in on the site of the DSB created by CRISPR-Cas9. This suggests FANCD2 plays an important role in regulating the insertion of new DNA at the cut site.
The researchers therefore believe FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.
“Also, since FANCD2 localizes to the site of Cas9 breaks, you can use FANCD2 to map where Cas9 is cutting in any cell type,” Dr Richardson said. “If you edit a population of cells and you want to know where the on- and off-target cuts are, you can just map where FANCD2 was found in the genome and you can find the cuts.”