Research ArticleMOLECULAR BIOLOGY

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

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Science Advances  24 Aug 2016:
Vol. 2, no. 8, e1600699
DOI: 10.1126/sciadv.1600699
  • Fig. 1 gRNA library construction using a semi-random primer.

    (A) Semi-random primer. Poly(A), polyadenylate. (B) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. (C) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. (D) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

  • Fig. 2 Guide sequences in the gRNA library.

    (A) Mass sequencing of the gRNA library. (B) An example of sequencing for 12 random clones. (C) An example of the BLAST search analysis of a guide sequence. The first guide sequence clone in Fig. 2A is shown as an example. A 20-bp guide sequence (red frame) is accompanied by a PAM (green frame). (D) Three different guide sequences derived from the same gene, the Ig heavy chain Cμ gene. (E) Features of the gRNA library. Percentages in the PAM graph were calculated among the guide sequences where their origins were identified. “Others” in the gRNA candidate graph indicates the sum of guide sequences of rRNA and PAM (−) mRNA.

  • Fig. 3 Functional validation of guide sequences.

    Three lentiviral clones specific to Cμ (Cμ guides 1, 2, and 3 in Fig. 2D) were transduced into the AID−/− sIgM (−) DT40 cell line. FACS profiles 2 weeks after transduction are shown with sIgM (−) gatings, which were used for FACS sorting (upper panels). The cDNA of the IgM gene from the sorted sIgM (−) cells is mapped together with the position of guide sequences, insertions, deletions, and mutations (lower panels). Detailed cDNA sequences around the guide sequences are shown at the bottom.

  • Fig. 4 Characterization and functional validation of the gRNA library.

    (A) Distribution of guide sequences on a chromosome. (B) Diversity of the gRNA library. Sequence reads per gene reflecting the transcriptomic landscape of the guide sequences (heatmap; shown with a scale bar). Guide sequence species per gene (circle graph). (C) Lentiviral transduction of gRNA library. Left: A FACS profile 2 weeks after transduction is shown with the sIgM (−) gating, which was used for FACS sorting. Right: The graph shows the total sequence reads in the library versus those in the sorted sIgM (−). Each dot represents a different gene. (D) IgM-specific guide sequences. Sequence reads specific to IgM (graph). Guide sequences mapped on IgM cDNA (map). Green and red bars represent guide sequences with forward and reverse orientations, respectively. (E) Deletions in the IgM cDNA in sorted sIgM (−). Left: The cDNA of the IgM gene from sorted sIgM (−) cells is shown, as well as the position of guide sequences, deletions, mutations, and exon borders. Right: Detailed sequences around breakpoints. Microhomologies in the reference sequences are highlighted in blue.

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