FIGURE SUMMARY
Title

Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish

Authors
Vanhooydonck, M., De Neef, E., De Saffel, H., Boel, A., Willaert, A., Callewaert, B., Claes, K.B.M.
Source
Full text @ Lab Anim (NY)
Fig. 1

Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR components with 100, 200, 400 or 800 pg Cas9 into the yolk of one-cell-stage zebrafish embryos. Left-hand y axes show EE (blue) and right-hand y axes show IF (gray). The bars and error bars represent the mean + s.e.m. of three biologically independent replicates (n = 3), where each replicate represents a pool of 20–30 injected embryos. Tukey’s multiple-comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2

Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR components into the cell or the yolk.Zebrafish embryos at the one-cell stage were injected with 200 pg (col1a2 c.918A>T), 400 pg (col1a2 c.1416A>C, col1a2 c.2093G>A) or 800 pg (atp6v1e1b c.634_636CGA>TGG, atp6v1e1b c.383T>C, brca2 c.6803G>A) Cas9 into the cell or the yolk. Left-hand y axes show EE (blue) and right-hand y axes show IF (gray). Bars and error bars represent the mean + s.e.m. of three biologically independent replicates (n = 3), where each replicate represents a pool of 20–30 injected embryos. Unpaired t-test.

Fig. 3

Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR templates, Alt-R HDR templates with silent guide-blocking variants or unmodified HDR templates.Zebrafish embryos at the one-cell stage were injected with 200 pg (col1a2 c.918A>T), 400 pg (col1a2 c.1416A>C, col1a2 c.2093G>A) or 800 pg (atp6v1e1b c.634_636CGA>TGG, atp6v1e1b c.383T>C, brca2 c.6803G>A) Cas9 into the yolk. Left-hand y axes show EE (blue) and right-hand y axes show IF (gray). The bars and error bars represent the mean + s.e.m. of three biologically independent replicates (n = 3), where each replicate represents a pool of 20–30 injected embryos. Dunnett’s multiple-comparisons test: *P < 0.05, **P < 0.01.

Fig. 4

Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR components and PE components. Zebrafish embryos at the one-cell stage were injected with 200 pg (col1a2 c.918A>T), 400 pg (col1a2 c.1416A>C, col1a2 c.2093G>A) or 800 pg (atp6v1e1b c.634_636CGA>TGG, atp6v1e1b c.383T>C, brca2 c.6803G>A) Cas9. EE and IF were compared with injection of PE components (RNP mixture containing 480 ng/µL pegRNA and 1,380 ng/µL PE2-His protein) into the yolk of one-cell-stage zebrafish embryos. Left-hand y axes show EE (blue) and right-hand y axes show IF (gray). The bars and error bars represent the mean + s.e.m. of three biologically independent replicates (n = 3), where each replicate represents a pool of 20–30 injected embryos. Unpaired two-tailed t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

F0 founder screen of 20 potential adult founder zebrafish generated by microinjection of HDR or PE components into the yolk of one-cell-stage zebrafish embryos. a, Results for zebrafish injected with Alt-R HDR components with 200 pg (col1a2 c.918A>T), 400 pg (col1a2 c.1416A>C, col1a2 c.2093G>A) or 800 pg (atp6v1e1b c.634_636CGA>TGG, atp6v1e1b c.383T>C, brca2 c.6803G>A) Cas9. b, Results for zebrafish injected with PE components (RNP mixture containing 480 ng/µL pegRNA and 1,380 ng/µL PE2-His protein).

Fig. 6

IF at top three predicted off-target locations. IF was determined for Alt-R HDR components with 200 pg (col1a2 c.918A>T), 400 pg (col1a2 c.1416A>C, col1a2 c.2093G>A) or 800 pg (atp6v1e1b c.634_636CGA>TGG, atp6v1e1b c.383T>C, brca2 c.6803G>A) Cas9 or PE components (RNP mixture containing 480 ng/µL pegRNA and 1,380 ng/µL PE2-His protein) injected into the yolk of one-cell-stage zebrafish embryos. The bars and error bars represent the mean + s.e.m. of three biologically independent replicates (n = 3), where each replicate represents a pool of 20–30 injected embryos. Unpaired two-tailed t-test: **P < 0.01, ***P < 0.001.

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 @ Lab Anim (NY)
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