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. Author manuscript; available in PMC: 2019 Aug 19.
Published in final edited form as: Drug Discov Today Technol. 2018 Feb 4;28:53–61. doi: 10.1016/j.ddtec.2018.01.001

Figure 3. Strategies for multiplexed CRISPR/Cas9-based genome editing.

Figure 3.

(A) In vivo pre-assembled RNP of Cas9 and multiple sgRNAs. (B) Introduction of Cas9 mRNA and multiple sgRNAs simultaneously. (C) Introduction of multiple single-sgRNA delivery plasmids simultaneously. (D) Delivery of Cas9, tracrRNA, and a crRNA array with a single plasmid. (E) Delivery of Cas9 and multiple sgRNA expression cassettes with a single plasmid. (F) Delivery of an artificial multi-sgRNA precursor, Cas9, and Csy4 with a single plasmid. sgRNAs are flanked by Csy4 binding sequences. Csy4 digests the precursor to release individual sgRNAs. (G) Delivery of an artificial multi-sgRNA precursor and Cas9 with a single plasmid. sgRNAs are flanked by HH and HDV ribozymes. The ribozymes digest the precursor to release individual sgRNAs. (H) Delivery of an artificial multi-sgRNA precursor and Cas9 with a single plasmid. sgRNAs are flanked by tRNAs. The endogenous tRNA processing machinery digests the precursor to release individual sgRNAs.