Mochii M et al. (2024), A CRISPR-Cas9-mediated versatile method for tar...

XB-ART-60446

Dev Biol 2024 Feb 01;506:42-51. doi: 10.1016/j.ydbio.2023.11.010.

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A CRISPR-Cas9-mediated versatile method for targeted integration of a fluorescent protein gene to visualize endogenous gene expression in Xenopus laevis.

Mochii M , Akizuki K , Ossaka H , Kagawa N , Umesono Y , Suzuki KT .

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Xenopus laevis is a widely used model organism in developmental and regeneration studies. Despite several reports regarding targeted integration techniques in Xenopus, there is still room for improvement of them, especially in creating reporter lines that rely on endogenous regulatory enhancers/promoters. We developed a CRISPR-Cas9-based simple method to efficiently introduce a fluorescent protein gene into 5' untranslated regions (5'UTRs) of target genes in Xenopus laevis. A donor plasmid DNA encoding an enhanced green fluorescent protein (eGFP) flanked by a genomic fragment ranging from 66 bp to 878 bp including target 5'UTR was co-injected into fertilized eggs with a single guide RNA and Cas9 protein. Injections for krt12.2.L, myod1.S, sox2.L or brevican.S resulted in embryos expressing eGFP fluorescence in a tissue-specific manner, recapitulating endogenous expression of target genes. Integrations of the donor DNA into the target regions were examined by genotyping PCR for the eGFP-expressing embryos. The rate of embryos expressing the specific eGFP varied from 2.1% to 13.2% depending on the target locus and length of the genomic fragment in the donor plasmids. Germline transmission of an integrated DNA was observed. This simple method provides a powerful tool for exploring gene expression and function in developmental and regeneration research in X. laevis.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: bcan krt12 krt12.2 myod1 sox2
GO keywords: embryo development
gRNAs referenced: bcan gRNA1 krt12.2 gRNA1 myod1 gRNA1 sox2 gRNA4

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	Graphical abstract
	Fig. 1. Targeted integration of egfp into krt12.2.L locus and expression of eGFP (A) Scheme of the donor DNA integration into 5′ UTR of the target gene. Light and dark blue boxes indicate 5′UTR. Brackets show homologous sequences containing promoter and 5′UTR. Red bars indicate sgRNA target sequences in the target locus and the donor plasmid. CDS, coding sequence of the target gene. DSB/NHEJ, DNA double strand break and non-homologous end joining. Indel, insertion and/or deletion. FW1/RV1 and FW2/RV2 indicate primer binding sites to amplify upstream and downstream junctions, respectively. (B–D) Representative images of a krt12.2.L:eGFP embryo at NF stage 45 injected with fgk537b-egfp. Clear eGFP fluorescence signal was observed in fin and gill. Signal was also found in pronephros and olfactory/oral region. (B), bright field image. (B′), fluorescence image of (B). (C, D), magnified partial images of (B′). g, gill. pn, pronephros. ol, olfactory pit. Asterisks indicate guts with autofluorescence. Bar in B, 1 mm. Bars in C and D, 500 μm. Representative images of tadpoles injected with fgk246b-egfp and fgk66b-egfp are shown in Figure S2 (E) Expression phenotypes of injected normal embryos were classified at around NF stage 40. Specific (blue), fin-and-gill-specific expression in both sides or either side. Partial (yellow), partial expression in fin and gill. Ectopic (gray), ectopic or ubiquitous expression. Rates of classified embryos are indicated as percentages in graphs. Donor DNAs and total numbers are indicated at top and bottom of graphs, respectively. Results of four (fgk567b-egfp and fgk246b-egfp) and five (fgk66b-egfp) independent experiments are combined. (F) PCR analysis of genomic DNAs from un-injected wild-type and embryos expressing eGFP with the specific pattern. Lanes 1–3, 4–6 and 7–9 indicate embryos injected with fgk537b-egfp, fgk264b-egfp and fgk66b-egfp, respectively. Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (krt12.2.L gFW and egfp RV1) and downstream (vector FW1 and krt12.2.L gRV) are shown in Supplemental Table 3. The predicted size of the upstream PCR fragment is 1062 bp if the donor is integrated in the forward direction with no indel. Predicted sizes of the downstream fragments are 849 bp, 552 bp and 312 bp for embryos injected with fgk537b-egfp, fgk264b-egfp and fgk66b-egfp, respectively. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S3.
	Fig. 2. Targeted integration of egfp into myod1.S locus and expression of eGFP. (A-C′) Representative images of myod1.S:egfp embryo and tadpole. (A–C), bright field images. (A′-C′), fluorescence images of (A–C). Clear eGFP signals were found in somitic muscles (A′, B′) and cranial muscles (C′). (A, A′, B, B′), lateral views. (C, C′), ventral view of head region. h, heart. Bars in A′ and B′, 1 mm. Bar in C′, 200 μm. Note that eGFP signal was not found in heart. (D) Expression phenotypes of injected normal embryos were classified at around NF stage 40. Specific, somite-specific expression in both sides or either side. Partial, partial expression in somites. Ectopic, ectopic or ubiquitous expression. Rates of classified embryos are indicated as percentage in or on left side of graph. Total number is shown below the graph. Combined data from nine experiments is shown. (E) PCR analysis of genomic DNAs from un-injected wild-type and embryos expressing the muscle-specific eGFP. Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (myod1.S gFW and egfp RV1) and downstream (vector FW2 and myod1.S gRV) are shown in Supplemental Table 3. Predicted size of the upstream PCR fragment is 1308 bp if the donor is integrated in the forward direction with no indel. Predicted size of the downstream fragment is 1601 bp. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S5.
	Fig. 3. Targeted integration of egfp into sox2.L locus and expression of eGFP (A-F) Representative images of sox2.L:egfp embryos. (A–C), bright field images. (A′-C′), fluorescence images of (A–C). (D–F), magnified partial images of (A′-C′). Specific eGFP signal was observed in brain (b), spinal cord (sc), olfactory pit (ol), optic vesicles (op) and otic vesicles (ot) in NF stage 32 embryo (A′, D). Signal was additionally observed in gill (g), lenses (le), lateral lines (la) in NF stage 41 (B′, E) and NF stage 45 (C′, F) embryos. (G) Expression phenotypes of injected normal embryos were classified at around NF stage 40. Specific, brain and spinal cord specific expression. Partial, partial expression in brain and/or spinal cord. Ectopic, ectopic and ubiquitous expression. Rates of classified embryos are indicated as percentage at left side of graph. Total numbers are indicated at bottom. Combined data from six experiments is shown. (H) PCR analysis of genomic DNAs from un-injected wild-type embryos and embryos expressing eGFP in the specific manner. Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (sox2.L gFW and egfp RV1) and downstream (vector FW2 and sox2.L gRV) are shown in Supplemental Table 3. Predicted size of the upstream PCR fragment is 1384 bp if the donor is integrated in the forward direction with no indel. Predicted size of the downstream fragment is 1275 bp. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S7. The large fragment in lane 4 of the downstream PCR could not be sequenced.
	Fig. 4. Targeted integration of egfp into bcan.S locus and expression of eGFP (A-E) Representative images of bcan.S:egfp embryos. (A, B), bright field images. (A′, B′), fluorescence images of (A, B). (C–E), magnified view of (A′, B′). Specific eGFP signal was observed in brain (b) and spinal cord (sc) in NF stage 45 embryos (A′, B′, C-E). Specific eEGFP signal in notochord (nc) was observed in some embryos (A′, D) but not in other embryos (B′, E). (F) Expression phenotypes of injected normal embryos were classified at around NF stage 43. Br/Sc + NC, specific expression in brain, spinal cord and notochord. Br/Sc, specific expression in brain and spinal cord but not in notochord. Ectopic, ectopic or ubiquitous expression. Rate of classified embryos is indicated as percentage in graph. Total numbers are indicated at bottom. Combined data from four experiments is shown. (G) PCR analysis of genomic DNAs from un-injected wild-type (wt) and tadpoles expressing eGFP in brain, spinal cord and notochord (1–3) and tadpoles expressing eGFP in brain and spinal cord but not in notochord (4, 5). Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (bcan.S gFW and egfp RV1) and downstream (vector FW1 and bcan.S gRV) are shown in Supplemental Table 3. Predicted size of the upstream PCR fragment is 1478 bp if the donor is integrated in the forward direction with no indel. Predicted size of the downstream fragment is 1046 bp. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S9.
	Fig. 5. Germline transmission of the donor DNA integrated in sox2.L locus. (A, A′) Representative photographs of F1 embryos at 3 days from crossing a wild-type female and a sox2.L:egfp male. Asterisks indicate eGFP-positive embryos. (B, B′) Representative images of eGFP-positive (upper) and -negative (lower) embryos at NF stage 40. (C, C′) Images of eGFP-positive tadpoles at NF stage 45. (A, B, C) bright field images. (A′, B′, C′) fluorescence images of (A, B, C). Bars, 1 mm. (D) PCR analysis of genomic DNAs from wild-type (wt) and eGFP-expressing embryos. Left and right gels show PCR fragments including upstream and downstream junctions, respectively.
	Fig S1. Sequence of krt12.2.L fragment in plasmid fgk537b-egfp. Gray and yellow highlighted letters indicate upstream and 5´UTR sequences of krt12.2.L. Underline indicates sgRNA target sequence with PAM sequence (red underline). Two arrows indicate primers for cloning of krt 12.2.L 264bp and 66bp fragments (fgk264b, fgk66b).
	Fig S2. Representative images of fgk264b-egfp and fgk66b-egfp embryos. (A, A’) Bright field and fluorescence images of fgf246b-egfp-injected embryo. (B, B’) Bright field and fluorescence images of fgf66b-egfp-injected embryo. g, gill. pr, pronephros. ol, olfactory pit. Bars, 1 mm.
	Fig S3. Sequences of the junction regions between the integrated donor DNAs and the targeted krt12.2.L locus. Partial sequences of 5´ UTRs, including upstream (upper) and downstream (lower) junctions, from embryos injected with fgk537b-egfp (1-3), fgk264b-egffp (4, 6) and fgk66b-egfp (7-9) are shown with the wild-type sequence. Downstream junction sequence from embryo 5 was not amplified by PCR (see Fig. 1F). Underline indicates the sgRNA target sequence with PAM sequence (red underline). Inserted nucleotides are indicated in red. Images show examples of sequence chromatograms for the junction regions. Indicated sequences are complementary to the 5´UTR sequence. Blue arrows indicate the deletion sites with the deleted sequences in parenthesis.
	Fig S4. Sequence of myod1.S fragment in plasmid myod1.S844b-egfp. Gray and yellow highlighted letters indicate upstream and 5´UTR sequences of myod1.S. Underline indicates sgRNA target sequence with PAM sequence (red underline).
	Fig S5. Sequences of the junction regions between the integrated donor DNA and the targeted myod1.S locus. Partial sequences of 5´ UTRs including upstream (upper) and downstream (lower) junctions from the four embryos expressing eGFP in the muscle-specific manner are shown with the wild-type sequence. Downstream junction sequences for embryo 1 and 3 were not amplified by PCR (see Fig. 2F). Underline indicates the sgRNA target sequence with PAM sequence (red underline). Inserted nucleotides are indicated in red. Images show examples of sequence chromatograms for the junction regions. Indicated sequences are complementary to the 5´UTR sequence. Blue arrows indicate the deletion sites with the deleted sequences in parenthesis.
	Fig S6. Sequence of sox2.L fragment in plasmid sox2.L787b-egfp. Gray and yellow highlighted letters indicate upstream and 5´UTR sequences of sox2.L. Underline indicates sgRNA target sequence with PAM sequence (red underline).
	Fig S7. Sequences of the junction regions between the integrated donor DNA and the targeted sox2.L locus. Partial sequences of 5´ UTRs including upstream (upper) and downstream (lower) junctions from the four embryos expressing eGFP in the specific manner are shown with the wild-type sequence. Downstream junction sequence for embryo 1 was not amplified by PCR (see Fig. 3F). Sequence of the downstream fragment from embryo 4 could not be determined (See Fig. 3F). Underline indicates the sgRNA target sequence with PAM sequence (red underline). Inserted nucleotides are indicated in red. Images show examples of sequence chromatograms for the junction regions. Indicated sequences are complementary to the 5´UTR sequence. Blue arrows indicate the deletion sites with the deleted sequences in parenthesis.
	Fig S8. Sequence of bcan.S fragment in plasmid bcan.S878b-egfp. Gray and yellow highlighted letters indicate upstream and 5´UTR sequences of bcan.S. Underline indicates sgRNA target sequence with PAM sequence (red underline).
	Fig S9. Sequences of the junction regions between the integrated donor DNA and the targeted bcan.S locus Partial sequences of 5´ UTRs including upstream (upper) and downstream (lower) junctions from the five embryos expressing eGFP in a specific manner are shown with the wild-type sequence. Downstream sequences of the major PCR products for tadpoles 2 and 5 were determined (See Fig. 4G). Underline indicates the sgRNA target sequence with PAM sequence (red underline). Inserted nucleotides are indicated in red. The 114 bp insertion in the tadpole 1 upstream junction is an inverted sequence of a part of bcan.S 5´UTR. The 198 bp insertion in tadpole 3 originated from the donor vector sequence. Images show examples of sequence chromatograms for the junction regions. Indicated sequences are complementary to the 5´UTR sequence. Blue arrows indicate the deletion sites with the deleted sequences in parenthesis.
	Fig S10. Comparison of the targeted integration in the myod1.S locus between myod1.S844b-egfp and myod1.S146b-egfp. Donor DNA with a truncated myod1.S fragment including only the 146b 5´UTR was constructed using a myod1.S146b FW primer (Supplemental table 1) and used for the targeted integration. (A, B) Representative bright field and fluorescence images of myod1.S844b-egfp-injected embryo (A, A’) and myod1.S146b-egfp-injected embryo (B, B’). Bars, 1 mm. (C) The eGFP expression ratio was compared between myod1.S844b-egfp and myod1.S146b-egfp injections. Results of two independent experiments are shown.