title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism.
abstract: The 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 describe 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.
authors: IFOM-FIRC Institute of Molecular Oncology Foundation, 20139 Milano, Italy. Email: hiroshi.arakawa@ifom.eu.
authors: Hiroshi
authors: 0000-0002-8103-4549
authors: H
authors: Arakawa
mesh_terms: D064113:CRISPR-Cas Systems; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D000072669:Gene Editing; D015723:Gene Library; D016678:Genome; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger
keywords: CRISPR; Cas9; Library; gRNA