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Genesis
2020 Jun 01;586:e23366. doi: 10.1002/dvg.23366.
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Simple embryo injection of long single-stranded donor templates with the CRISPR/Cas9 system leads to homology-directed repair in Xenopus tropicalis and Xenopus laevis.
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We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).
EY022954 NIH HHS , K01DK101618 NIH HHS , Sharon Stewart Aniridia Trust, Vision for Tomorrow, K01 DK101618 NIDDK NIH HHS , EY022954 National Institutes of Health, K01DK101618 National Institutes of Health
FIGURE 1
Rescuing tyr mutant of X. tropicalis. (a) Design of the donor long single-stranded DNA (lssDNA). The top section shows nucleotide sequences of a part of the lssDNA and the corresponding tyr mutant locus where lowercase letters (red) are silent mutations and “xxx….x” (magenta) are mutations in the Target 1 site. The underlined italic region is a newly made KpnI site in the Target 2 sgRNA site. The CRISPR target sites were described previously (Nakayama et al., 2013) and shown in blue with PAM sequences in orange. The bottom section shows schematically the genomic region (gray) of tyr mutants where “x” (magenta) refers to uncharacterized mutations. Target 2 site shown in the blue square is used to induce a DSB by CRISPR. The lssDNA is shown in green. (b) Representative rescued phenotypes, where white arrows show examples of rescued melanophores. The scale for scoring is from – to +++ as described in the text. (c) The left section shows the schematic strategy of genotyping via KpnI digestion. Tail clipping of Embryos 1–3 shown in (b) was done and tissues were lysed for genomic PCR followed by KpnI digestion. The right section shows the resulting band patterns with (KpnI) or without (non) restriction digestion. The white asterisk shows the position of doublet bands (492 bp + 505 bp) created by KpnI digestion. CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; DSB, double-strand break
FIGURE 2
Germline transmission of HDR-mediated recombined loci in X. tropicalis. (a) Four F0 partially rescued frogs were tested for germline transmission, showing that Females 1 and 3 laid 25–32% pigmented eggs. (b) Genotyping of albino and pigmented eggs from Female 1 by KpnI digestion. The red asterisk highlights the doublet bands as seen in Figure 1c. (c) F1 embryos generated by crossing F0 Female 1 with an albino male. From white eggs, albino embryos were seen as expected (e.g., bottom), whereas from pigmented eggs 50% formed normally pigmented embryos (e.g., top) and 50% formed lightly pigmented (shown by white arrows) embryos (e.g., middle). (d) Molecular profiles of the recombined loci. At the top, locations of SNPs (shown in red) present in some of the parental frogs used for experiments are shown where the 3′ end of the long single-stranded DNA (lssDNA) is denoted by 1 and the 5′ end by 898 (note that the lssDNA is the antisense strand but numbers are shown in the sense strand). Immediately below this representation of genomic SNP locations is a sequence alignment showing an example of a non-HDR-recombined tyr locus, cloned from pigmented eggs, which was repaired by NHEJ at the Target 2 locus. The middle sequence alignment is an example of an HDR-mediated recombined locus, where several SNPs are not rescued, suggesting that only the middle part of the lssDNA (shown with green bar) was used for recombination. The bottom sequence alignment is another example of an HDR-mediated recombined locus, where recombination occurred at the targeted site but consistent with recombination from only the 5′ half of the lssDNA (green bar). Note that “Female 2” shown here had a developmental defect (bending to the left) that we sometimes see even in wild-type frogs possibly due to suboptimal husbandry (too high density) while raising tadpoles but not related to the HDR-procedure. HDR, homology-directed repair; NHEJ, nonhomologous end joining; SNP, single-nucleotide polymorphism
FIGURE 3
Genotyping of F1 X. tropicalis embryos for potential random genomic insertion of the long single-stranded DNA (lssDNA) used for rescue of the albino mutant. The left section shows the schematic strategy of genotyping via KpnI digestion, which would create two restriction bands (see green and yellow asterisks) when integration of the lssDNA (green bar where red asterisks indicate point mutations generating the KpnI site) occurs. The right section shows the resulting band patterns with (KpnI) or without (non) restriction digestion. Albino Embryos 1–4* were derived from white eggs, and 5–8* from pigmented eggs. The pigmented Embryo 9* is used as a positive control for KpnI digestion. DNA samples 4*, 8*, and 9* are from the bottom, middle, and top embryos, respectively, shown in Figure 2c. No evidence was seen for random insertion of lssDNA among tested embryos shown here
FIGURE 4
HDR-mediated seamless integration of sfGFP in the slc45a2.L locus of X. laevis. (a) The schematic diagram of HDR-mediated knock-in of sfGFP in the slc45a2.L locus with three long single-stranded DNA (lssDNA) constructs having different length HAs (orange lines). (b) Schematic diagram of the strategy for genomic PCR used to genotype F0 animals. The results show bands of the expected sizes in an embryo that has undergone HDR (and shows expected fluorescence). (c) F0 animals show sfGFP-positive cells in the epidermis as expected. Left panel, Stage 36 embryo, here outlined for clarity, with the anterior (A), posterior (P), dorsal (D), and ventral (V) sides labeled. One can clearly see sfGFP positive cells (e.g., red arrowheads) even with relatively high background fluorescence from yolk cells at this stage. Middle panel, Stage 50 embryo at high magnification to show part of the dorsolateral trunk region. One can see several sfGFP-positive nonpigmented cells (e.g., red arrowheads). Right panel, a high magnification view of the leg skin of a froglet, by which stage one can see patches of groups of sfGFP-positive cells (e.g., red arrowheads). (d) Left panel, an sfGFP-positive F1 froglet (left) with a sibling control froglet (right), outlined for clarity. Right panel, the genomic PCR for genotyping of an sfGFP-positive F1 froglet (HDR) and a sibling control (wt), showing the expected sizes of bands for integrated (#) and nonintegrated alleles (*). Diagrams are not drawn to scale. HDR, homology-directed repair; HAs, homology arms; sfGFP, Superfolder green fluorescent protein; SNP, single-nucleotide polymorphism
