Fishing for Outstanding Abilities
(October 31st, 2017) Faced with the poor annotation of the chicken genome, molecular biologist Hiroshi Arakawa developed a method to disrupt gene expression via the CRISPR/Cas9 technology, without prior knowledge of target DNA sequences.
CRISPR/Cas9 is a sequence-specific endonuclease that can cleave any locus in the genome. It is commonly used selectively to inactivate genes via sequence-specific guide RNAs. Using messenger RNA, Hiroshi Arakawa created a random guide RNA library that can be used as a tool for forward genetic screening. “I plan to use such libraries to treasure hunt outstanding species-specific abilities. I would like to uncover, for example, the genetic basis of the extraordinary regeneration ability of planaria, the cancer resistance of naked mole rats or the extended life span of red sea urchins,” he explained. “With my method, even some forgotten species might be converted into attractive genetic models.” Since 2011, Arakawa has been a Principal Investigator and staff scientist with Marco Foiani at the IFOM, the Molecular Oncology Institute of the Italian Foundation for Cancer Research in Milan, Italy.
“After trying to develop the method for half a year, I suddenly had the key idea when taking a stroll near Sempione Park, Milan. This was the starting point of this project. However, at first, I did not believe that my approach would work,” Arakawa revealed his misgivings. Even as a very experienced researcher, initially, he had to become familiar with several new methods and techniques, such as CRISPR, very short-range PCR and large-scale plasmid libraries. As a result, he now has been successful with the following process: he has synthesised complementary DNA from the messenger RNA of a chicken B cell line, using special semi-random primers for reverse transcription. These primers contained two crucial elements: a so-called Protospacer Adjacent Motif (PAM) complementary sequence, which is required for target binding by Cas9, and a random 20-mer guide sequence, which defines the target sequence to be modified. Subsequently, he isolated the reverse transcribed guide sequences with special restriction enzymes and used them to build a library.
“The library’s complexity and diversity were much greater than I had expected,” Arakawa observed. The guide sequences represented over 90 per cent of the annotated chicken genes and multiple guide sequences were generated from the same gene. When he transduced a surface IgM-expressing chicken B cell line with lentiviral clones that contained guide sequences targeting the immunoglobulin heavy chain gene Cµ, he was able to disrupt surface IgM expression. With further analyses, he proved that the library could be used for the generation of deletions, insertions and mutations within genomic as well as messenger RNA sequences. “The published method has two weak points. It is biased toward highly expressed genes and the knockout efficiency is low,” he noted. “These weak points have now been improved. In addition, I’m currently developing another new technology for forward genetic screening.”
Before he came to Milan, Arakawa worked for several years as a Senior Research Fellow at the Helmholtz Zentrum in Munich and as a postdoc at the Heinrich Pette Institute in Hamburg, Germany. He had obtained his PhD from Kyoto University in 1996. In these posts, he became expert in immunoglobulin gene conversion, somatic hypermutation and their use for artificial evolution.
Moving to Milan was an easy decision. His employer offered him a two-year contract, an excellent scientific environment and scientific freedom. “IFOM is one of the best institutes in Italy. They have the most advanced technology in oncology. Moreover, the people here are relaxed and easy-going and the food is nice. However, I had to get used to the slightly different time management in Italy. Japanese tend to be stricter in this regard,” the scientist observed.
One or two further papers or inventions would give him a chance to get more funding for his research and additional employees, he said. The demands are high. “In Italy, funding for life science is focussed on cancer research or human genetic diseases. Funding agencies request a high impact factor of more than 30 over the previous five years as either the first or final author,” he explained. As IFOM is opening new branches in Asian countries, should the opportunity arise, he would move to an IFOM institute in Japan.
“There are at least two large frontiers in biology: one is the ‘dark matter’ of the human genome, which occupies more than 90 per cent of the genome and whose functions are poorly known. Another is poorly characterised non-model species. I believe that with my forward genetic approaches I could exploit this goldmine of unknown genetic treasures,” he observed.