The Universal sgRNA (UgRNA) promotes high efficiency targeted integration.
(a) Universal sgRNA (UgRNA) sequence. Cas9 PAM underlined. (b) Schematic of targeting using the UgRNA to direct CRISPR/Cas cutting in the donor vector. The genomic sgRNA target site sequence in noto exon 2 is shown in bold green. The sequence of the UgRNA in the donor vector is shown in bold black. PAM sequences are underlined, and Cas9 cut sites are indicated with red arrow. The 24 bp noto homology arm in the donor vector is in green; since it lacks the last 3 base pairs and PAM sequence found at the genomic noto target site it is not recognized by the noto sgRNA. (c) Frequency of injected embryos displaying RFP expression in the notochord compared to total number of injected embryos following targeting using the noto sgRNA, UgRNA, and UgRNA-24bp-2A-tagRFP-CAAX vector shown in (b).
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