FIGURE

Fig. 2

ID

ZDB-FIG-250220-13

Publication

Kim et al., 2024 - CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos

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Fig. 2

Sequential cloning strategy used to generate one- and two-color CRISPR-Cas13d systems. (A) Donor plasmid supplying the backbone vector contains the following features: Origin of replication (Ori), Ampicillin resistance (AmpR), ubiquitous promoter (CAG), and a membrane-localized RFP reporter (memRFP), as well as a multiple cloning site. Relevant restriction sites are shown. (B-D) Donor plasmid was digested with indicated restriction enzymes and ligated iteratively to insert three guide RNAs (gRNAs). Three variations of the gRNA plasmid were created: one containing memRFP for a two-color system (D), and two others containing split GFP(11) reporters that are either nuclear (nucGFP(11)) or membrane localized (3xmemGFP(11)) for a one-color system (C). Donor plasmid was also separately digested with indicated restriction enzymes and ligated with respective inserts to create two variations of the Cas13d plasmid: one containing a split GFP reporter (GFP(1–10)) for the one-color system (C), and the other containing a Citrine reporter for the two-color system (D). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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Reprinted from Developmental Biology, , Kim, M., Hutchins, E.J., CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos, , Copyright (2024) with permission from Elsevier. Full text @ Dev. Biol.