Fig. 4

A comparison of morpholino- and CRISPR-Cas13d-mediated PAX7 knockdown approaches. (A-D) Representative maximum intensity projections of HH9+ chick embryos electroporated with morpholino (MO) or CRISPR-Cas13d reagents and immunostained for PAX7 (magenta). (A) MO knockdown embryos were bilaterally electroporated with a control morpholino (Ctrl MO) and an RFP reporter (left), and Pax7 MO and a GFP reporter (right). (B) Representative transverse cross-section of embryo shown in (A). (C) One-color CRISPR-Cas13d knockdown embryos were electroporated with Cas13d + Control gRNA plasmids (left), and Cas13d + Pax7 gRNA plasmids (right), using split GFP fluorescence to indicate co-expression of both plasmids. Cyan outlines the area of PAX7+ cells, which is used to calculate the area of neural crest migration. (D) Representative transverse cross-section of embryo shown in (C). (E) Quantification of the neural crest migration defect. Each data point represents the relative area of neural crest migration away from the midline, indicated by PAX7+ cells, detected on the right side of an embryo (PAX7 knockdown) compared to the left side of the same embryo (contralateral control). A statistically significant reduction in PAX7+ migration area is observed with MO-mediated knockdown of PAX7 (80.0 ± 4.1% of PAX7+ cell migration area compared to control; n = 11 embryos). ∗, p = 0.0029, one-sample Wilcoxon signed-rank test. Similarly, a statistically significant reduction in PAX7+ migration area is also observed with CRISPR-Cas13d-mediated knockdown of PAX7 (75.5 ± 4.5% of PAX7+ cell migration area compared to control; n = 13 embryos). ∗, p = 0.0012, one-sample Wilcoxon signed-rank test. Notably, the defects observed in neural crest migration for the two modes of knockdown are not different. ns, nonsignificant; p = 0.2066, Mann-Whitney test. Scale bar, 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)