Abstract
While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in most species, except for well-established model organisms, such as human, mouse, or yeast. A guide RNA (gRNA) library using the clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system can be used for the phenotypic screening of uncharacterized genes by forward genetics. The construction of a gRNA library usually requires an abundance of chemically synthesized oligos designed from annotated genes; if one wants to convert mRNA into gRNA without prior knowledge of the target DNA sequences, the major challenges are finding the sequences flanking the protospacer adjacent motif (PAM) and cutting out the 20-bp fragment. Recently, I developed a molecular biology-based technique to convert mRNA into a gRNA library (Arakawa, 2016) (Figure 1). Here I describe the detailed protocol of how to construct a gRNA library from mRNA. Figure 1.A method to convert mRNA into a gRNA library construction ( Sanjana et al., 2014 ). The scheme of the method is summarized. Each step of D-O is described in detail in the Procedure. Bg, BglII; Xb, XbaI; Bs, BsmBI; Aa, AatII. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2.