A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism | Zendy

Hiroshi Arakawa | Zendy

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A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

Author(s) -

Hiroshi Arakawa

Publication year - 2016

Publication title -

science advances

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 5.928

H-Index - 146

ISSN - 2375-2548

DOI - 10.1126/sciadv.1600699

Subject(s) - crispr , guide rna , computational biology , messenger rna , rna editing , rna , genome editing , cas9 , primer (cosmetics) , organism , biology , subgenomic mrna , computer science , genetics , chemistry , gene , organic chemistry

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.

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