Supplementary Information:
CRISPR-mediated activation of latent HIV-1 expression
Prajit Limsirichai1, *, Thomas Gaj2, * and David V. Schaffer2, 3, 4, 5
1
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley,
California 94720, USA
2
Department of Chemical and Biomolecular Engineering, University of California, Berkeley,
Berkeley, California 94720, USA
3
Department of Bioengineering, University of California, Berkeley, Berkeley, California 94720,
USA
4
Department of Cell and Molecular Biology, University of California, Berkeley, Berkeley,
California 94720, USA
5
Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, California
94720, USA
*
These authors contributed equally to this work
Correspondence and requests for materials should be addressed to D.V.S.
(schaffer@berkeley.edu)
ACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTA
CCACACACAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGGGCCAGGGGACA
GATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGA
TAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCAT
GGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGC
ATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCG
AGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCG
GGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGT
ACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGG
AACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC
CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGG
AAAATCTCTAGCAG
Supplementary Fig. 1. Long-terminal repeat (LTR) sequence from the HIV-1 subtype B
strain HXB2 (NCBI accession number: K03455.1). sgRNA target sites are highlighted yellow.
Positions of variation between the reference LTR and the sequence used for the design and
validation of the sgRNA are indicated in red.
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Supplementary Fig. 2. “Tiling” multiple sgRNAs across the HIV-1 LTR promoter does not
dramatically enhance the efficiency of dCas9-VP64-mediated gene activation. Fold-increase
in the percentage of EGFP positive HEK293T cells after transfection with 20 ng of EGFP
reporter plasmid, 90 ng of dCas9-VP64 expression vector, and 90 ng of sgRNA expression
vector. For tiling experiments, the amount of sgRNA-encoded DNA was kept constant so that
each transfection contained 90 ng of total sgRNA. Data normalized to EGFP expression in cells
transfected with sgRNA 3. Only sgRNAs 4 and 7 induced a significant increase in gene
activation compared to sgRNA 3. See Fig. 1d for the activity of each individual sgRNA. EGFP
expression was measured 48 hr after transfection. Error bars indicate s.d. (n = 3; *p-value < 0.05;
Student’s t-test).
3
Supplementary Fig. 3. Tiling multiple sgRNAs across the HIV-1 LTR promoter does not
significantly increase the efficiency of SAM-mediated gene activation. Fold-increase in the
percentage of EGFP positive HEK293T cells after transfection with 20 ng of EGFP reporter
plasmid, 60 ng of dCas9-VP64 expression vector, 60 ng of MS2-p65-HSF1 expression vector,
and 60 ng of sgRNA expression vector. For tiling experiments, the amount of sgRNA-encoded
DNA was kept constant so that each transfection contained 60 ng of total sgRNA vector. Data
normalized to EGFP expression in cells transfected with sgRNA 3. See Fig. 1d for the activity of
each individual sgRNA. EGFP expression was measured 48 hr after transfection. Error bars
indicate s.d. (n = 3; p-values from Student’s t-test).
4
Supplementary Fig. 4. Co-transfection of SAM with a library of non-specific sgRNA does
not lead to a significant increase in gene activation from the HIV-1 LTR. Fold-increase in
the percentage of EGFP positive HEK293T cells after transfection with 20 ng of EGFP reporter
plasmid, 60 ng of dCas9-VP64 expression vector, 60 ng of MS2-p65-HSF1 expression vector,
and 60 ng of an empty sgRNA expression cassette, or a library1 of sgRNAs designed to target the
proximal promoter of each human RefSeq coding isoform. EGFP expression was normalized to
cells transfected with the empty sgRNA expression cassette and measured 48 hr after
transfection. Error bars indicate s.d. (n = 3; p-value from Student’s t-test).
5
Supplementary Fig. 5. Tumor necrosis factor alpha (TNF-α) stimulation of J-Lat 9.2 and JLat 10.6 cells. Distribution histograms generated by flow cytometry indicating the percentage of
GFP positive naïve J-Lat 9.2 (upper) and J-Lat 10.6 (lower) cells following stimulation with
increasing amounts of TNF-α. GFP expression was measured 24 hr after treatment.
6
Supplementary Fig. 6. Prostratin treatment of J-Lat cells expressing SAM is not as
effective at stimulating viral gene expression as combination treatment with SAHA.
Percentage of GFP positive J-Lat 9.2 (upper) and J-Lat 10.6 (lower) cells stably expressing SAM
with sgRNAs 1 to 7 and treated with either 4 µM SAHA only, 2 µM prostratin only, or 4 µM
SAHA plus 2 µM prostratin. “Empty” indicates J-Lat cells expressing SAM with an empty
sgRNA expression cassette. Dotted line indicates the percentage of GFP positive naïve J-Lat
cells after treatment with 20 ng/µL of TNF-α. Cells were treated 17 days after infection, and GFP
expression was measured 24 hr after treatment. Error bars indicate standard deviation (n = 3).
7
5’ LTR sgRNA 1
CACCGACAAGATATCCTTGATCTG
3’ LTR sgRNA 1
AAACCAGATCAAGGATATCTTGTC
5’ LTR sgRNA 2
CACCGATTGACAGAACTACACACC
3’ LTR sgRNA 2
AAACGGTGTGTAGTTCTGTCAATC
5’ LTR sgRNA 3
CACCGTCAGATATCCACTGACCTT
3’ LTR sgRNA 3
AAACAAGGTCAGTGGATATCTGAC
5’ LTR sgRNA 4
CACCGCTACAAGGGACTTTCCGCT
3’ LTR sgRNA 4
AAACAGCGGAAAGTCCCTTGTAGC
5’ LTR sgRNA 5
CACCGGCGTGGCCTGGGCGGGACT
3’ LTR sgRNA 5
AAACAGTCCCGCCCAGGCCACGCC
5’ LTR sgRNA 6
CACCGGTTAGACCAGATCTGAGCC
3’ LTR sgRNA 6
AAACGGCTCAGATCTGGTCTAACC
5’ LTR sgRNA 7
CACCGCCCGTCTGTTGTGTGACTC
3’ LTR sgRNA 7
AAACGAGTCACACAACAGACGGGC
Supplementary Fig. 7. Primer sequences used to construct the sgRNA used in this study.
Bases complementary to the LTR are yellow highlighted.
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REFERENCES
1.
Konermann S, et al. Genome-scale transcriptional activation by an engineered CRISPRCas9 complex. Nature 517, 583-588 (2015).
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