Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells
- Laboratory of Molecular Epigenomics and Chromatin Organization, Genome Institute of Singapore, Singapore
- Cardiovascular Disease Translational Research Programme, Yong Loo Lin School of Medicine, Singapore
- Laboratory of Systems Biology and Data Analytics, Genome Institute of Singapore, Singapore
- Laboratory of RNA Genomics and Structure, Genome Institute of Singapore, Singapore
- Center for Genome Diagnostics, Genome Institute of Singapore, Singapore
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, Singapore
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Precision Medicine Translational Research Programme, Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Figures
Figure 1 with 7 supplements
Computational search for candidate GSH.
(A) Schematic representation of the computational workflow for defining candidate GSH. (B) CIRCOS plot summarising computational search results. Ring 1: chromosome ideograms; ring 2: orange bars …
Figure 1—figure supplement 1
Hi-C profile for GSH candidate 1.
Screenshots of Hi-C interaction matrices from H1 hESC for targeted GSH candidate locus on chromosome 1 (113339961-113340514). TADs are indicated by the ‘pyramids’ of high interaction observed in the …
Figure 1—figure supplement 2
Hi-C profile for GSH candidate 2a.
As in Figure 1—figure supplement 1 but for GSH targeted GSH candidate locus on chromosome 2 (128912721-128914814).
Figure 1—figure supplement 3
Hi-C profile for GSH candidate 2c.
As in Figure 1—figure supplement 1 but for GSH targeted GSH candidate locus on chromosome 2 (128932307-128935799).
Figure 1—figure supplement 4
Hi-C profile for GSH candidate 4.
As in Figure 1—figure supplement 1 but for GSH targeted GSH candidate locus on chromosome 4 (17373361-17374159).
Figure 1—figure supplement 5
Hi-C profile for GSH candidate 6.
As in Figure 1—figure supplement 1 but for GSH targeted GSH candidate locus on chromosome 6 (15727241-15727490).
Figure 1—figure supplement 6
Hi-C profile for GSH candidate 18.
As in Figure 1—figure supplement 1 but for GSH targeted GSH candidate locus on chromosome 18 (56534775-56536439).
Figure 1—figure supplement 7
Hi-C profile for GSH candidate 19.
As in Figure 1—figure supplement 1 but for GSH targeted GSH candidate locus on chromosome 19 (5400761-5402139).
Figure 2 with 9 supplements
GSH targeting and transcriptomic analysis of H1 hESC.
(A) Schematic representation of CRISPR/Cas9 plasmid (pMIA3) and homology directed repair donor (pMIA4.721) used for targeting with functional components annotated. (B) Schematic of integrated …
Figure 2—figure supplement 1
PCR gel images of junction PCR and wild type allele PCR reactions for screened clones.
Figure 2—figure supplement 2
Off-target screening for Pansio-1.
Sanger sequencing traces of the top five predicted gRNA off-target sites for un-targeted wild type H1 & H9 hESC and Pansio-1 GSH targeted clones. All sequencing traces are ordered as follows: H1 WT, …
Figure 2—figure supplement 3
Off-target screening for Olônne-18.
As in Figure 2—figure supplement 2, but for Olônne-18.
Figure 2—figure supplement 4
Off-target screening for Keppel-19.
As in Figure 2—figure supplement 2, but for Keppel-19.
Figure 2—figure supplement 5
Transcriptomic analysis of GSH targeted H9 hESC.
(A) Log2-FC of mRNA expression levels against un-targeted H9 hESC samples for the nearest genes of Pansio-1, Olônne-18, and Keppel-19 candidate GSH. Evaluated samples: H9=un-targeted hESC, …
Figure 2—figure supplement 6
Representative images of metaphase spreads used for karyotyping the GSH targeted H1 and H9 cell lines.
Figure 2—figure supplement 7
Representative images of haematoxylin and eosin stained teratoma from Pansio-1, Olônne-18, and Keppel-19 H1 cells.
Examples of mesodermal, endodermal, and ectodermal tissues are presented. Scale bar 100 µm.
Figure 2—figure supplement 8
Inducible expression from GSH integrated cassette.
(A) Schematic representation of ‘all-in-one’ inducible transposon donor construct (pMIA10.7). (B) Schematic of landing pad construct with integrated ‘all-in-one’ inducible cassette. (C) …
Figure 2—figure supplement 9
Schematic representation of the pMIA10.53-Clover donor plasmid.
Figure 3 with 2 supplements
Integration and validation of transgene expression in GSH targeted H1 hESC and their differentiated progeny.
(A) Schematic representation of integrase expression construct (pMIA22) and transposon donor construct (pMIA10.5). (B) Schematic of landing pad construct with integrated Clover transgene. (C) …
Figure 3—figure supplement 1
Integration and validation of transgene expression in GSH targeted H9 hESC and their differentiated progeny.
(A) Representative immunofluorescence images of Clover-integrated GSH H9 cells. DAPI = nuclear staining with 4′,6-diamidino-2-phenylindole, Clover = fluorescence from Clover transgene, …
Figure 3—figure supplement 2
Representative images of H1 Pansio-1 line immunofluorescence staining with AF594 secondary antibody alone.
DAPI = nuclear staining with 4′,6-diamidino-2-phenylindole, Clover = fluorescence from Clover transgene, AF594=staining with secondary antibody alone, Overlay = overlay of the three imaged channels.
Figure 4 with 1 supplement
Representative images of Pansio-1 H9 cells differentiated to neuronal, hepatic, and cardiac cell types.
Staining for respective lineage markers TUJ1, HNF4α and cTnT and isotype controls is shown as well as nuclear staining with HOECHST and the channel for Clover-transgene. Images are composites of 61 …
Figure 4—figure supplement 1
Bar plots representing the mean ratio of positive cells from the high content imaging analysis.
Error bars indicate 95% confidence intervals. Individual data points shown in pink.
Tables
Table 1
Coordinates of candidate GSH, their associated active chromosome regions & housekeeping gene, and BLAT score against the most similar region.
| Chromosome | Start | End | Width | Active region start | Active region end | Housekeeping gene | BLAT-score |
|---|---|---|---|---|---|---|---|
| 1 | 113289036 | 113289342 | 307 | 113000001 | 114000000 | HIPK1 | 0.21 |
| 1 | 113314841 | 113318369 | 3529 | 113000001 | 114000000 | HIPK1 | 0.18 |
| 1 * | 113339961 | 113340514 | 554 | 113000001 | 114000000 | HIPK1 | 0.27 |
| 2 * | 128912721 | 128914814 | 2094 | 128000001 | 129000000 | UGGT1 | 0.08 |
| 2 | 128918961 | 128919839 | 879 | 128000001 | 129000000 | UGGT1 | 0.16 |
| 2* | 128932307 | 128935799 | 3493 | 128000001 | 129000000 | UGGT1 | 0.15 |
| 2 | 128963272 | 128965759 | 2488 | 128000001 | 129000000 | UGGT1 | 0.44 |
| 2 | 208992998 | 208997459 | 4462 | 208000001 | 209000000 | PIKFYVE | 0.28 |
| 4* | 17373361 | 17374159 | 799 | 17000001 | 18000000 | MED28 | 0.04 |
| 5 | 131058585 | 131058947 | 363 | 131000001 | 132000000 | FNIP1 | 0.41 |
| 5 | 148753741 | 148757219 | 3479 | 148000001 | 149000000 | FBXO38 | 0.10 |
| 6 * | 15727241 | 15727490 | 250 | 15000001 | 16000000 | DTNBP1 | 0.47 |
| 7 | 4314741 | 4315279 | 539 | 4000001 | 5000000 | FOXK1 | 0.15 |
| 7 | 4321017 | 4323839 | 2823 | 4000001 | 5000000 | FOXK1 | 0.09 |
| 7 | 4328040 | 4329659 | 1620 | 4000001 | 5000000 | FOXK1 | 0.21 |
| 7 | 4353504 | 4354219 | 716 | 4000001 | 5000000 | FOXK1 | 0.32 |
| 7 | 4454808 | 4456201 | 1394 | 4000001 | 5000000 | FOXK1 | 0.17 |
| 8 | 23945241 | 23945819 | 579 | 23000001 | 24000000 | R3HCC1 | 0.34 |
| 8 | 23986981 | 23988319 | 1339 | 23000001 | 24000000 | R3HCC1 | 0.06 |
| 8 | 23999628 | 24001194 | 1567 | 23000001 | 24000000 | R3HCC1 | 0.02 |
| 18 | 56339813 | 56340245 | 433 | 56000001 | 57000000 | TXNL1 | 0.06 |
| 18 | 56396821 | 56397319 | 499 | 56000001 | 57000000 | TXNL1 | 0.07 |
| 18 | 56410681 | 56411039 | 359 | 56000001 | 57000000 | TXNL1 | 0.14 |
| 18 * | 56534775 | 56536439 | 1665 | 56000001 | 57000000 | TXNL1 | 0.16 |
| 19 * | 5400761 | 5402139 | 1379 | 5000001 | 6000000 | SAFB | 0.18 |
-
*
= GSH shortlisted for in vitro validation.
Author response table 1
| Olônne-18 GSH targeted H1 hESC |
| Keppel-19 GSH targeted H1 hESC |
| Control targeted H1 hESC (CRISPR and HDR of an expression cassette at a non-GSH locus) |
| Independent untargeted H1 hESC |
Additional files
-
Supplementary file 1
Output of safe and active searches and their overlap.
(1) Coordinates that pass safe site filters. (2) Regions that pass active filters. (3) Overlap of safe sites and active regions.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp1-v2.xlsx
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Supplementary file 2
Oligos, primers and antibodies used, and details of beta cell differentiation.
(1) gRNAs used for targeting. (2) Primers used for plasmid construction. (3) Primers used for junction PCR and WT allele screening. (4) Primers used for predicted off-target screening. (5) Antibodies used for immunofluorescence staining and flow cytometry. (6) Details for pancreatic differentiation.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp2-v2.xlsx
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Supplementary file 3
GSH targeted hESC screening.
(1) Summary of GSH targeted hESC clones screening. (2) ddPCR copy number analysis.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp3-v2.xlsx
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Supplementary file 4
Source data for qPCR results.
(1) qPCR data source file for Figure 2C. (2) qPCR data source file for Figure 2–figure supplement 5A.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp4-v2.xlsx
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Supplementary file 5
RNAseq analysis of GSH targeted H1 clones.
(1) Differentially expressed genes |logFC|≥1 and FDR ≤0.01. (2) Functional enrichment analysis results from g:GOSt.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp5-v2.xlsx
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Supplementary file 6
RNAseq analysis of GSH targeted H9 clones.
(1) Differentially expressed genes |logFC|≥1 and FDR ≤0.01. (2) Functional enrichment analysis results from g:GOSt.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp6-v2.xlsx
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Supplementary file 7
HCI data analysis source file for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp7-v2.xlsx
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Supplementary file 8
Percentage of FITC-A positive cells from H1 Pansio inducible cells source file for Figure 2—figure supplement 8.
- https://cdn.elifesciences.org/articles/79592/elife-79592-supp8-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79592/elife-79592-mdarchecklist1-v2.docx
