Boosting targeted genome editing using the hei-tag
- Centre for Organismal Studies (COS), Heidelberg University, Germany
- Heidelberg Biosciences International Graduate School (HBIGS), Germany
- Institute of Pharmacology, Heidelberg University, Germany
- DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, Germany
Figures
Figure 1 with 2 supplements
heiCas9 exhibits outstanding bi-allelic targeting activity in fish.
Phenotypic range and quantification of OlOca2 T1, T2, and DrOca2 T1, T2 sgRNAs/Cas9 variant and sgRNA/Cas9 protein complex (ribonucleoprotein [RNP])-mediated loss of pigmentation in medaka (a–d) and …
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Figure 1—source data 1
Raw data for quantifications shown in Figure 1d and g.
- https://cdn.elifesciences.org/articles/70558/elife-70558-fig1-data1-v3.csv
Figure 1—figure supplement 1
Identification of the hei-tag.
Comparison of OlOca2 knock-out efficiency (quantification of eye pigmentation) using a permutation screen of peptide domains (nuclear localization signals [NLSs], Myc-tag, amino acid linkers) …
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Figure 1—figure supplement 1—source data 1
Raw data for quantifications shown in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/70558/elife-70558-fig1-figsupp1-data1-v3.csv
Figure 1—figure supplement 2
Survival and abnormality rate of Cas9 mRNA and ribonucleoprotein (RNP) injections.
Percentage of dead, abnormal (e.g. delayed development or malformation), and properly developed injected embryos. Only properly developed embryos were included for analysis. n, total number of …
Figure 2 with 1 supplement
Increased knock-out activity and reduced allele variance in heiCas9 crispants.
Multiplexed injections with 15ng/µl mRNA of zCas9 or heiCas9 (red) mRNA and 12.5ng/µl per sgRNA targeting exonic sequences in oculocutaneous albinism type 2 (oca2; OlOca2 T2), the start codons of …
Figure 2—figure supplement 1
Mode of editing of all modified alleles.
Relative abundance of Illumina reads categorized by mode of editing among all modified alleles per replicate, locus (OlOca2, OlRx2, OlRx3, OlCryaa) and Cas9 mRNA employed (zCas9, heiCas9). …
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Figure 2—figure supplement 1—source data 1
Raw data for barplots shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/70558/elife-70558-fig2-figsupp1-data1-v3.xlsx
Figure 3 with 1 supplement
heiCas9 consistently exhibits high genome editing efficiency in mammalian cells.
Mouse SW10 cells were co-transfected with MmPrx crRNA and mRNAs of JDS246-Cas9, GeneArt CRISPR nuclease, and heiCas9, respectively. Genome editing efficiency was assessed by Tracking of Indels by …
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Figure 3—source data 1
Raw data for scatter plot shown in Figure 3.
- https://cdn.elifesciences.org/articles/70558/elife-70558-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Representative indel spectrum for each Cas9 mRNA used in the cell culture assay.
Indel spectrum diagram obtained from Tracking of Indels by Decomposition (TIDE) (red bargraphs) and Inference of CRISPR Editing (ICE) (blue bargraphs) analyses following JDS246-Cas9 mRNA, GeneArt …
Figure 4 with 1 supplement
heiBE4-Gam mediates highly efficient cytosine-to-thymine (C-to-T) transitions in medaka embryos.
Phenotypic range and quantification of heiBE4-Gam-mediated C-to-T transitions in medaka embryos. (a) Categories of typically observed loss-of-pigmentation phenotypes in oca2 editants. The observed …
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Figure 4—source data 1
Raw data for quantifications shown in Figure 4a.
- https://cdn.elifesciences.org/articles/70558/elife-70558-fig4-data1-v3.csv
Figure 4—figure supplement 1
Increased cytosine-to-thymine (C-to-T) transition in medaka embryo pools injected with heiBE4-Gam.
(a) Schematic representation of base editing window in OlOca2 T1 target site. (b–c) Sanger sequencing quantifications (EditR; Kluesner et al., 2018) of pools of five randomly picked embryos injected …
Tables
Table 1
Primer sequences used for Cas9 variant cloning.
Restriction enzyme sites used for cloning are indicated in italics (AgeI in the forward primer, XbaI in the reverse primer), underscored sequence, binding to Cas9 open reading frame (ORF). F, …
| Primer name | Primer sequences in 5’–3’ |
|---|---|
| MFO-Cas9_fwd | AATTTACCGGTTTACCATGGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGAGGAAGCGGACCACCTCCCAAGAGGCCCAGGCTGGACCTCGAGGATAAAAAGTATTCTATTGGTTTAG |
| MIS-Cas9_fwd | AATTTACCGGTTTACCATGGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGTATCCACGGAGTCCCAGCAGCCGCTCCAAAGAAGAAGCGTAAGGTAGATAAAAAGTATTCTATTGGTTTAG |
| MSF-Cas9_fwd | AATTTACCGGTTTACCATGGAGCAGAAGCTGATCAGCGAGGAGGACCTGATGGCTCCAAAGAAGAAGCGTAAGGTAGGAGGAAGCGGAGATAAAAAGTATTCTATTGGTTTAG |
| OMF-Cas9_fwd | AATTTACCGGTTTACCATGCCACCTCCCAAGAGGCCCAGGCTGGACCTCGAGGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGAGGAAGCGGAGATAAAAAGTATTCTATTGGTTTAG |
| SMF-Cas9_fwd | AATTTACCGGTTTACCATGGCTCCAAAGAAGAAGCGTAAGGTACTCGAGGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGAGGAAGCGGAGATAAAAAGTATTCTATTGGTTTAG |
| Cas9-O_rev | AATTTTCTAGATTAGTCCAGCCTGGGCCTCTTGGGAGGAGGGGATCCGTCACCCCCAAGCTGTGAC |
| Cas9-S_rev | AATTTTCTAGATTAATCTACCTTACGCTTCTTCTTTGGAGCAGCGGATCCGTCACCCCCAAGCTGTGACA |
| myc-Cas9_fwd | AATTTACCGGTCAAACATGGAGCAGAAGCTGATCAGCGAGGAGGACCTGATGGCCCCAAAGAAGAAGCGGAAGGTC |
| myc-Cas9_rev | AATTTTCTAGATTACTTTTTCTTTTTTGCCTGGCCGGC |
Table 2
Primer sequences used for BE4-Gam and heiBE4-Gam cloning.
| Primer name | Primer sequences in 5’–3’ |
|---|---|
| pCS2+_backbone_fwd | GCCTCTAGAACTATAGTGAGTCG |
| pCS2+_backbone_rev | ATGGGATCCTGCAAAAAGAACAAG |
| hei-tag_fragment_fwd | CTTGTTCTTTTTGCAGGATCCCATTTACCATGGAGCAGAAGCTG |
| hei-tag_fragment_rev | GCTGGTTTAGCCTCGAGGTCCAGCCTGG |
| Gam_Mu-APOBEC1-Cas9n_fragment_fwd | GACCTCGAGGCTAAACCAGCAAAACGTATCAAG |
| Gam_Mu-APOBEC1-Cas9n_fragment_rev | CTAGGGCCTTGAGAAGTGTC |
| Cas9n-UGI_fragment_fwd | GACACTTCTCAAGGCCCTAG |
| Cas9n-UGI_fragment_rev | CAGAGTCACCCCCAAGCTG |
| 2xUGI-oNLS_fwd | CAGCTTGGGGGTGACTCTG |
| 2xUGI-oNLS_rev | CGACTCACTATAGTTCTAGAGGCTTAGTCCAGCCTGGGCCTCTTGGGAGGGGGAGAACCACCAGAGAGC |
Table 3
Locus-specific primers with 5’ partial illumina adapter sequences.
Locus-specific primers with Illumina adapter sequence underscored.
| Primer name | Primer sequences in 5’–3’ |
|---|---|
| oca2_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTTAGAGTGGTATGGAGAACTGT |
| oca2_R | GACTGGAGTTCAGACGTGTGCTCTTCCGATCTATGGTCCTCACATCAGCAGC |
| cryaa_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGCCATTTGCTTGTGTGTCA |
| cryaa_R | GACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGTCTAGGAGGATGGGGCAG |
| rx2_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGAGGCACAAGAACTATTTGTTGATC |
| rx2_R | GACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGGGCTCCGTTAACTTTGGG |
| rx3_F | ACACTCTTTCCCTACACGACGCTCTTCCGATCTATGCAAACCAAGAAAGCGCC |
| rx3_R | GACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGGGATTTCTCAAAGGCCCG |
Additional files
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Supplementary file 1
Nucleotide and translated amino acid sequence of heiCas9.
- https://cdn.elifesciences.org/articles/70558/elife-70558-supp1-v3.docx
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Supplementary file 2
Sequences of peptide tags fused to mammalian SpCas9 (Figure 1, Supplementary file 1).
M, cMyc-tag; O, optimized nuclear localization signal (NLS) (Inoue et al., 2016); S, SV40 NLS (Kalderon et al., 1984); Xl, bipartite Xenopus laevis nucleoplasmin NLS (Dingwall et al., 1988).
- https://cdn.elifesciences.org/articles/70558/elife-70558-supp2-v3.docx
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Supplementary file 3
Allele variants and abundance in OlOca2, rx2, rx3, and cryaa.
Sequences and abundance of all locus-mapped reads per replicate (pool) and locus (OlOca2, rx2, rx3, cryaa) of multiplexing with either zCas9 or heiCas9 mRNA injections (Figure 2). Sequences of allele variants (with more than 100 reads) displayed.
- https://cdn.elifesciences.org/articles/70558/elife-70558-supp3-v3.pdf
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70558/elife-70558-transrepform1-v3.docx
