High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

Abstract

The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.

Keywords: CRISPR/Cas9; F0; Homology-directed repair (HDR); Non-mosaic; Xenopus laevis; Xenopus tropicalis.

Fig. 1.

Comparison of CRISPR/Cas9 methods in Xenopus oocytes and embryos. (A) Schematic comparing the oocyte isolation/host transfer method (left) with direct embryo injection of CRISPR/Cas9 (right). The generation time for X. laevis is 12-16 months and for X. tropicalis 4-10 months. (B) Overall efficiency of indel generation in F0. The numbers of viable tadpoles from injected oocytes and the percentage of embryos with single indel mutations are shown for the host transfer and embryo injection methods. There is no mosaicism in embryos derived from host transfer method (number of different indel mutations per embryo=1). (C) Examples of genotyping analysis of indel mutations of X. tropicalis F0 embryos. The target sequence is underlined and the protospacer adjacent motif (PAM) is highlighted in blue characters.

Fig. 2.

Comparative analysis of HDR in Xenopus oocytes and embryos. (A) The procedure for precisely inserting a 2×FLAG tag into the ctnnb1 gene through an HDR-mediated recombination event. (B) Representative western blot of single X. laevis embryos obtained by oocyte injection/host transfer. In the bottom image the green bands represent β-catenin protein and the red band represents the FLAG tag. (C) Representative western blot with of X. laevis embryos derived by embryo injection. (D) Whole-mount immunostaining of epithelial tissues shows expression of the FLAG tag in the same pattern as β-catenin protein in oocyte-injected samples, as expected for heterozygous embryos, and in just a few cells in embryo injected samples, as expected for mosaic embryos. Scale bars: 250 μm (red); 30 μm (white). (E) Real-time PCR (top) shows the stability (mean±s.d. from three independent experiments) of sgRNA in oocytes and western blot (bottom) demonstrates the decay of 600 pg Cas9 protein injected into oocytes and embryos of X. laevis. (F) Sequencing of 20 (for 2×FLAG) or 24 (for V5) clones from the oocyte-injected embryo shows successful knock-in of 2×FLAG in one allele of ctnnb1 and of the V5 epitope in one allele of vangl2.S. PAM sequences are marked in blue; green indicates mismatched nucleotide from the RO; red indicates epitope. No conclusions were drawn from lane 7 owing to damage to the blot. (G) Treatment of oocytes with the DNA ligase IV inhibitor SCR-7 (5 µM) increases the efficiency of successful HDR-mediated knock-in. Representative western blots are shown for ctnnb1.S:2×HA with or without SCR-7 treatment. (H) The overall efficiency of HDR events in F0 tadpoles. Xt, X. tropicalis; Xl, X. laevis.

Fig. 3.

Germline transmission of X. tropicalis Tg(ctnnb1:2×FLAG) knock-in. Representative images showing successful FLAG epitope tagging of β-catenin protein in four embryos (red arrows). The embryo marked with the blue arrow is stained with DAPI only (no primary antibody).