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

Sci Adv. 2016 Aug 24;2(8):e1600699. doi: 10.1126/sciadv.1600699. eCollection 2016 Aug.

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.

Keywords: CRISPR; Cas9; Library; gRNA.

MeSH terms

  • CRISPR-Cas Systems*
  • Computational Biology
  • DNA, Complementary / genetics
  • Gene Editing*
  • Gene Library
  • Genome
  • RNA, Guide / genetics*
  • RNA, Messenger / genetics*

Substances

  • DNA, Complementary
  • RNA, Guide
  • RNA, Messenger