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Volume 7, Issue 4 (Suppl)
J Biotechnol Biomater, an open access journal
ISSN: 2155-952X
Bio America 2017
October 19-20, 2017
October 19-20, 2017 | New York, USA
18
th
Biotechnology Congress
Amethod to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
Hiroshi Arakawa
IFOM–FIRC Institute of Molecular Oncology Foundation, Italy
T
he clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful
tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one
must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally
making gRNA. I have described a method to construct a gRNA library via molecular biology techniques without relying on
bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing
a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes
to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it
applicable to any species.
Biography
Hiroshi Arakawa studied at Kyoto University (Kyoto, Japan), where he obtained his diploma and Ph.D in Molecular Biology in Hideo Yamagishi’s laboratory. Following post-
doctoral studies in Jean-Marie Buerstedde's laboratory in Heinrich-Pette-Institut (Hamburg, Germany), he worked as a Senior Research Fellow in Jean-Marie Buerstedde's
laboratory in Helmholtz Center Munich (Munich, Germany). He moved to IFOM (Milan, Italy) as a staff scientist in 2011. He has so far studied the molecular mechanism of
immunoglobulin gene conversion and somatic hypermutation, and their application to artificial evolution system. He has recently invented a method to convert mRNA into
a gRNA library, which can be applied to forward genetic screening in any species.
hiroshi.arakawa@ifom.euHiroshi Arakawa, J Biotechnol Biomater 2017, 7:4 (Suppl)
DOI: 10.4172/2155-952X-C1-079