FIGURE

Figure 4

ID

ZDB-FIG-210915-87

Publication

de Vrieze et al., 2021 - Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

Other Figures

All Figure Page

Back to All Figure Page

Figure 4

Proposed strategy for the efficient generation of knock-in zebrafish. Step 1 entails the verification of sgRNA-Cas9 efficiency at creating genomic lesions using high resolution melting (HRM) analysis. Step 2 concerns the injection of sgRNA-Cas9 ribonucleoprotein complexes and the asymmetric antisense oligonucleotide template with the variant of interest. Next generation sequencing (NGS) is recommended to determine knock-in efficiency. In the situation of low knock-in efficiency, knockdown of non-homologous end-joining protein Ku70 (by co-delivery of an morpholino antisense oligonucleotide (PMO), targeting xrcc6) may offer a means to improve knock-in efficiency. Finally, injected embryos with sufficient knock-in reads can be raised to adults and screened for founders with the variant of interest in the germline (step 3). The white boxes in step 1 and step 2 described the key components injected into the zebrafish zygote. A detailed protocol can be found in Supplemental Document S1.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Int. J. Mol. Sci.