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. 2021 Dec 6;10:e75050. doi: 10.7554/eLife.75050

Figure 1. Cloning-free single-copy CRISPR/Cas9-mediated knock-in (KI) lines in medaka.

(A) Schematic diagram of cloning-free CRISPR knock-in strategy. The injection mix consists of three components, a single-guide RNA (sgRNA) targeting the gene of interest, Cas9-mSA mRNA, and the PCR-amplified donor fragment containing short homology arms on both ends (30–40 bp) and the fluorescent protein of interest with no ATG and no stop codon. Note that the 5′ ends of the PCR donor fragment are biotinylated (Btn). The mix is injected in one-cell staged medaka embryos and the injected fishes are screened for potential in-frame integrations mediated by homology-directed repair (HDR). (B) eGFP-cbx1b F1 CRISPR KI line stage 40 medaka embryos. eGFP-Cbx1b labels all nuclei and is thus an ubiquitous nuclear marker. n > 10 embryos. Scale bar = 100 µm (C) mScarlet-pcna F1 CRISPR KI line stage 40 medaka embryos. mScarlet-Pcna labels exclusively cycling cells. n > 10 embryos. Scale bar = 100 µm. (D) mNG-myosinhc F1 CRISPR KI line stage 40 medaka embryos. mNG-Myosinhc labels exclusively muscle cells located in the myotome tissue of medaka embryos. n > 10 embryos. Scale bar = 100 µm.

Figure 1.

Figure 1—figure supplement 1. Schematic representation of mNeonGreen-myosinhc tagging strategy.

Figure 1—figure supplement 1.

PCR amplification of mNeonGreen-HAtag-Linker (green, orange, and purple) using primers homologous to the extremity of the insert and containing flanking sequence corresponding to the homology arms (blue) for insertion just downstream the ATG of myosinhc gene (yellow) following Cas9/sgRNA DNA-induced cut. The PCR primers contain 5′ end Biotins (Btn). Bold letters denote the single-guide RNA (sgRNA) PAM, underlined the sequence upstream the PAM recognized by the sgRNA, blue letters the sequence homologous between the myosinhc locus and the PCR donor, dashed green for mNeonGreen indicates it is not to scale.
Figure 1—figure supplement 2. Alignment of whole genome sequencing (WGS) reads to fluorescent protein sequences.

Figure 1—figure supplement 2.

(A) eGFP integration in cbx1b locus. Paired-end sequenced reads from eGFP-cbx1b F1 embryos, two biological replicates (gDNA1 and gDNA2) from the same F0 founder are shown in the upper and lower panels, coloured in grey for concordant mappings and coloured in turquoise/purple for inter-chromosomal paired-ends with one mate mapped to chr19 (left panel) and one mate mapped to eGFP (right panel). The predicted integration site is shown as a vertical dashed line with soft-clipped reads (shown as coloured bases) to the left and right of the integration site. Above the sequenced reads are the basepair-level coverage histogram. The results indicate that eGFP integrated only at the cbx1b locus genome wide as we could not detect eGFP reads anchored anywhere else in the genome. Purple (right)/turquoise (left) bars are of particular importance as they represent reads that span both the eGFP and the endogenous cbx1b locus. The two dark grey bars represent unpaired mate, mate not mapped, or otherwise unknown status. The mapping quality of these two reads is 0 and therefore they most likely represent false mappings. The red bars are anomalous mappings that either deviate from the expected insert size or paired-end mapping orientation. Coverage of eGFP-cbx1b_gDNA1 is 20.4× and for eGFP-cbx1b_gDNA2 is 23.6×. (B) mScarlet integration in pcna locus. Paired-end sequenced reads from mScarlet-pcna F1 embryos, coloured in grey for concordant mappings and coloured in turquoise/green for inter-chromosomal paired-ends with one mate mapped to chr9 (left panel) and one mate mapped to mScarlet (right panel). The predicted integration site is shown as a vertical dashed line with soft-clipped reads (shown as coloured bases) to the left and right of the integration site. Above the sequenced reads are the basepair-level coverage histogram. The results indicate that mScarlet integrated only at the pcna locus genome wide as we could not detect mScarlet reads anchored anywhere else in the genome. Green (right)/turquoise (left) bars are of particular importance as they represent reads that span both mScarlet and the endogenous pcna locus. The red bars are anomalous mappings that either deviate from the expected insert size or paired-end mapping orientation. The one purple bar is flagged as a possible inter-chromosomal read between chromosomes 9 and 17. The mapping quality of this read is 0 which most likely means it is a false mapping. Coverage of mScarlet-pcna is 14.4×. (C) mNeonGreen integration in myosinhc locus: paired-end sequenced reads from mNG-myosinhc F1 embryos, coloured in grey for concordant mappings and coloured in dark grey/purple for inter-chromosomal paired-ends with one mate mapped to chr8 (left panel) and one mate mapped to mNeonGreen (right panel). The predicted integration site is shown as a vertical dashed line with soft-clipped reads (shown as coloured bases) to the left and right of the integration site. Above the sequenced reads are the basepair-level coverage histogram. Paired-end analysis yielded a second, weakly supported partial insertion of mNeonGreen at chr12:9083923 (not shown) which could not be confirmed by subsequent PCR, suggesting that this is a false-positive prediction or a rare insertion of very low frequency. The results argue that mNeonGreen integrated only at the myosinhc locus genome wide. Purple (right)/dark grey (left) bars are of particular importance as they represent reads that span both mNeonGreen and the endogenous myosinhc locus. The red bars are anomalous mappings that either deviate from the expected insert size or paired-end mapping orientation. Coverage of mNG-myosinhc is 14.5×.
Figure 1—figure supplement 3. Sanger sequencing of F1 lines confirms single-copy in-frame fusion proteins.

Figure 1—figure supplement 3.

(A) Sanger sequencing read from eGFP-cbx1b F1 fin-clipped adult shows scarless integration of eGFP into the cbx1b locus in both 5′ and 3′ junctions. Consensus coverage sequence is located at the top most panel in blue and grey. Any mismatch will be flagged by a black colour. There are no mismatches present. Blue line on top of the chromatograph peaks (A = green, T = red, C = blue, G = black) represent the quality of sequenced bases. (B) Sanger sequencing reads from mScarlet-pcna F1 fin-clipped adult show scarless integration of mScarlet into the pcna locus at the 3′ junction. While the 5′ junction shows correct in-frame fusion of endogenous pcna to mScarlet, we were able to detect a partial duplication of the 5′ homology arm with a small insertion that occurred 22 bp upstream of the start codon of endogenous pcna (for details see Materials and methods). Consensus coverage sequence is located at the top most panel in blue and grey. Any mismatch will be flagged by a black colour. There are no mismatches present. Blue line on top of the chromatograph peaks (A = green, T = red, C = blue, G = black) represents the quality of sequenced bases. (C) Sanger sequencing reads from mNG-myosinhc F1 fin-clipped adult show scarless integration of mNeonGreen into the myosinhc locus in both 5′ and 3′ junctions. Consensus coverage sequence is located at the top most panel in blue and grey. Any mismatch will be flagged by a black colour. There are no mismatches present. Blue line on top of the chromatograph peaks (A = green, T = red, C = blue, G = black) represents the quality of sequenced bases.