Using magnetic beads, magnets, and a robot, a team of scientists have optimized the production of gene-editing tools that target and edit specific sequences of an organism’s genome.
The new method quickly produces a large number of enzymes that target specific DNA sequences. The enzymes, called transcription activator-like effector nucleases (TALENs), are made by combining proteins that bind to specific DNA segments and an enzyme, called a nuclease, that cuts through double-stranded DNA. These TALENs can introduce new genetic material to the targeted location in the genome. While TALENs hold much promise for gene therapy applications and for studying basic gene function, it has been difficult and expensive to create large numbers of TALENs at once.
“We can now rapidly and reliably knock out large numbers of genes, which is important for studying the functions of individual genes as well as large gene networks,” said Jeffry Sander, a co-senior author and a fellow in J. Keith Joung’s laboratory at Massachusetts General Hospital.
Adam Bogdanove, a plant pathologist at Iowa State University who was not involved in the study, said the FLASH TALENs method gives scientists a way to “systematically identify what mismatches can be tolerated.” The high-throughput technique will also show "what the implications will be for trying to edit individual genes without affecting other parts of the genome,” he said.
To test the FLASH method, Sander and his collaborators constructed 48 pair of TALENs targeted to a gene sequence that codes for a fluorescing protein. All 48 pairs became active when placed in human cells. The team also made FLASH TALEN pairs targeted to 96 human genes involved in cancer or epigenetics. The team’s TALENs introduced targeted modifications into 84 of those genes.
“Because we used an architecture of TALENs that has worked efficiently in nematodes, rats, zebrafish, and human cells, we think FLASH-assembled TALENs will also be active and work well in other organisms,” said Joung, a Massachusetts General Hospital pathologist and co-senior author. Although the FLASH method produced effective TALENS with this particular architecture, TALENs based on other architectures might not necessarily have the same success, he noted.
Also, the results show that longer TALENs had less toxic effects on the cells in which they were inserted. The finding suggests that shorter TALENs may bind to and alter unintended gene sites more easily. These mismatches happen with one of the fundamental pieces of a TALEN, but, in nature, cells typically can tolerate the gene coding mistakes. Scientists aren’t yet sure how these mismatches affect gene-editing laboratory techniques.
1. Reyon, D., S. Q. Tsai, C. Khayter, J. A. Foden, J. D. Sander, and J. K. Joung. 2012. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotech advance online publication(April).