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Biochem Biophys Res Commun
2021 Mar 05;543:50-55. doi: 10.1016/j.bbrc.2020.11.038.
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Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis.
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The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.
Fig. 1. Efficiency of knock-down (KD) via microinjection was improved by low-temperature incubation of embryos. (A) Representative mutants and uninjected control. The knock-down embryos were classified into 4 groups: excellent, no or very few pigments were seen; good, pigmentation present, but less than half that in the wild-type; partial, pigments were decreased, but still more than half the volume of the wild type; none, eyes and retinas were fully pigmented. Scale bar: 1 mm. (B) Frequency of pigmentation level of knock-down embryos and uninjected control (n = 44–59 in each condition. Each sample number is shown in Supplementary Table S2). Uninjected controls were kept at 12 °C for 6 h and reared at 20 °C thereafter. We carried out the same experiments twice and the number of embryos was classified in each experiment with representative results shown. Other results are shown in Supplementary Fig. S1. ∗P < 0.01, NS (not significant), Fisher’s exact test with Holm correction.
Fig. 2. Indel mutation rates of each knock-down embryo confirmed by analysis using the ICE algorithm. The graph shows the indel mutation percentage of samples (n = 6–13) that was successfully aligned with the control sequence obtained from a WT embryo in ICE analysis and its R-Squared was greater than 0.9. Bars indicate averages. ∗P < 0.01, NS (not significant), Tukey-Kramer test. Representative data of detected indel mutation patterns and distributions in the edited sample are shown in Supplementary Fig. S2.
Fig. 3. HDR-mediated knock-in (KI) using the CRISPR/Cas9 system and lssDNA. (A) Schematic diagram of HDR-mediated knock-in into the exon of tyr. S. Blue boxes and yellow boxes indicate untranslated regions and coding domain sequences, respectively. (B) Representative fluorescence images of (left column) heads, (center column) trunks, and (right column) tails of (first row) an uninjected control embryo, (second row) an embryo injected with lssDNA-only, and (third to fifth row) knock-in mutants. The embryos were classified into 4 groups according to the expression level of GFP: excellent, GFP expressed widely throughout the body; good, GFP expression was seen sparsely throughout the body; partial, GFP expression was seen sparsely in specific parts of the whole body; none, no GFP expression was seen. Arrowheads and asterisks indicate strong GFP signals and autofluorescence, respectively. White dashed lines indicate outlines of embryos. Scale bar: 1 mm. (C) Higher magnification image of trunk of an “excellent” knock-in embryo showing widespread GFP expression. Note that there are many GFP-expressing cells other than those with strong GFP expression indicated by arrowheads. Scale bar: 100 μm. (D) Frequency of GFP expression level in 3 groups (incubated at 12 °C for 0, 6, and 24 h, respectively), injected lssDNA-only, and uninjected control group (n = 37–51 in each condition. Sample numbers are shown in Supplementary Table S2). Embryos injected with lssDNA-only and uninjected controls were kept at 12 °C for 6 h and reared at 20 °C thereafter. The duration of the low-temperature incubation did not affect GFP expression lssDNA-only injected groups (Supplementary Fig. S3). We carried out the same experiments 3 times and representative results are shown. Other results are shown in Supplementary Fig. S4. ∗P < 0.01, NS (not significant), Fisher’s exact test with Holm correction. (E) Schematic diagram (top) of the strategy for genomic PCR used for genotyping and representative results (bottom). (F) Sequencing of 8 (for upstream) and 6 (for downstream) knock-in embryos and uninjected control. The tyr gRNA sequences are marked in blue; both ends of the CMV-acgfp cassette are marked in red; and PAM sequences are marked in green. The entire sequences of these samples are shown in Supplementary Fig. S5.
Fig. 4. Quantification of knock-in (KI) efficiency in individuals by flow cytometry. (A) Schematic drawing of the procedure for flow cytometry. (B) The proportion of GFP+ 7-AAD- cells in total 7-AAD- cells of knock-in and control embryos (n = 8 in each condition). Around 50,000 events from each embryo are acquired by FACS Canto II. ∗P < 0.05, NS (not significant), Tukey-Kramer test.
Fig. S1. Frequency of pigmentation level of knock-down (KD) embryos and uninjected control (n=30-34 in each condition. Sample numbers are shown in Supplementary Table S2). *P<0.01, NS (not significant), Fisher's exact test with Holm correction.
