Target Health Blog

Dr. Yoshizumi Ishino - CRISPR

February 11, 2019

History of Medicine

Graphic credit: by marius walter - Own work, CC BY-SA 4.0,

Yoshizumi Ishino is a Japanese molecular biologist, known for his discovering the DNA sequence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Ishino has contributed to the development of enzymology and nucleic acids research all his life. In 1987, he discovered the DNA sequence of CRISPR, which is a basis of a biotechnology known as CRISPR-Cas9 that effectively edits genes.

Yoshizumi Ishino graduated from the faculty of Pharmaceutical Science, Osaka University, Japan, with a BS (1981) and MS (1983). His research was on structure and function of restriction endonucleases. He then received his PhD in 1986 at the Research Institute for Microbial Diseases, Osaka University, based on his research on the E. coli DNA ligase. Yoshi later did a postdoc with Prof. Dieter Soll at Yale University (1987-1989) conducting research on translation-related enzymes. After completing his PhD, Yoshi returned to Japan to join the Bioproducts Development Center of Takara Shuzo, Japan, where he rose to senior research scientist in the Biotechnology Research Laboratories. Dr. Ishino later joined the Biomolecular Engineering Research Institute (BERI), a national project funded by the Japanese Government (METI) and 18 major companies in the Biotechnology field. At BERI, he managed a research group on nucleic acids-related enzymes. In 2002, he was appointed a full professor of protein chemistry and engineering in the Department of Genetic Resources Technology, Kyushu University.

In 1990, Yoshi started his research on DNA replication in Archaea. Archaea constitute a domain of single-celled microorganisms. Archaea are prokaryotes, meaning they have no cell nucleus. Yoshi cloned the gene encoding a family B DNA polymerase from the hyperthermophilic archaeon, Pyrococcus furiosus. Furthermore, he identified two family B DNA polymerase genes in Pyrodictium occultum; but more interestingly, he discovered an archaea specific DNA polymerase from P. furiosus. This enzyme was designated PolD and a new family (family D) was proposed for this new DNA polymerase after it was found that it is conserved in Euryarchaeota. Yoshi expanded his research area to recombinational repair in Archaea after 1996. In this field, he discovered an archaea-specific Holliday junction resolvase named Hjc and later also discovered a novel enzyme from P. furiosus which he named Hef (helicase-associated endonuclease for fork structured DNA). It is interesting that the human ortholog of the archaeal Hef is FANCM, which is implicated in the genetic disease Fanconi Anemia.

Currently, Yoshi focuses his research not only on enzymes of DNA replication and recombinational repair in archaea, but also on their evolutionary relationships with counterpart proteins in the eukaryotic domain. In 2002, he became a professor at Kyushu University. Since October 2013, he has been a member of the NASA Astrobiology Institute, University of Illinois at Urbana - Champaign. In 2017, he was honored with the AMED Award, Japan Agency for Medical Research and Development (AMED). In 2018, he was awarded the JSBBA Award, Japan Society for Bioscience, Biotechnology and Agrochemistry (JSBBA)

In 1987, Dr. Yoshizumi Ishino and his research team accidentally cloned part of a CRISPR together with the iap gene, the target of interest. The organization of the repeats was unusual because repeated sequences are typically arranged consecutively along DNA. They studied the relation of “iap“ to the bacterium E. coli. The function of the interrupted clustered repeats was not known at the time. In 1993, researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in this bacterium. These researchers recognized the diversity of the DR-intervening sequences among different strains of M. tuberculosis and used this property to design a typing method that was named spoligotyping, which is still in use today. In 2019, Dr. Ruud Jansen and his scientific team continue CRISPR research. Repeats were observed in the archaeal organisms of Haloferax and Haloarcula species, and their function was studied by Francisco Mojica at the University of Alicante in Spain. Although his hypothesis turned out to be wrong, Mojica's supervisor surmised at the time that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division because plasmids and chromosomes with identical repeat arrays could not coexist in Haloferax volcanii.

Transcription of the interrupted repeats was also noted for the first time. By 2000, Mojica performed a survey of scientific literature and one of his students performed a search in published genomes with a program devised by himself. They identified interrupted repeats in 20 species of microbes as belonging to the same family. In 2002, Tang, et al. showed evidence that CRISPR repeat regions from the genome of Archaeoglobus fulgidus were transcribed into long RNA molecules that were subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units. A major addition to the understanding of CRISPR came with Dr. Ruud Jansen's observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Four cas genes (cas 1 - 4) were initially recognized. The Cas proteins showed helicase and nuclease motifs, suggesting a role in the dynamic structure of the CRISPR loci. The three major components of a CRISPR locus are: cas genes, a leader sequence, and a repeat-spacer array. Several CRISPRs with similar sequences can be present in a single genome, only one of which is associated with cas genes.

In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids. In effect, the spacers are fragments of DNA gathered from viruses that previously tried to attack the cell. The source of the spacers was a sign that the CRISPR/cas system could have a role in adaptive immunity in bacteria. All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals. The first publication proposing a role of CRISPR-Cas in microbial immunity, by the researchers at the University of Alicante, predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the RNA interference system used by eukaryotic cells. Koonin and colleagues extended this RNA interference hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins. Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007, the first experimental evidence that CRISPR was an adaptive immune system was published. A CRISPR region in Streptococcus thermophilus acquired spacers from the DNA of an infecting bacteriophage. The researchers manipulated the resistance of S. thermophilus to phage by adding and deleting spacers whose sequence matched those found in the tested phages.

In 2008, Brouns and Van der Oost identified a complex of Cas proteins (called Cascade) that in E. coli cut the CRISPR RNA precursor within the repeats into mature spacer-containing RNA molecules (crRNA), which remained bound to the protein complex. Moreover, it was found that Cascade, crRNA and a helicase/nuclease (Cas3) were required to provide a bacterial host with immunity against infection by a DNA virus. By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting dsDNA. That year Marraffini and Sontheimer confirmed that a CRISPR sequence of S. epidermidis targeted DNA and not RNA to prevent conjugation. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in Pyrococcus furiosus. A 2010 study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophilus. A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III CRISPR-RNA complex in the accompanying diagram.

CRISPR/Cas genome editing techniques have many potential applications, including medicine and crop seed enhancement. The use of CRISPR/Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015. Bioethical concerns have been raised about the prospect of using CRISPR for germline editing.

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