Expression and hydroxyurea-triggered induction of EGFP upon CRISPR/Cas9-mediated integration into the γ-globin gene of K562 cells
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To knock-in an EGFP cassette into the γ-globin genes of K562 cells via CRISPR/Cas9, and to assess expression and hydroxyurea (HU)-mediated induction of the targeted EGFP transgene.
The EGFP cassettes were specifically knocked into the Gγ gene. EGFP expression was detected in the targeted cell population and isolated clones. Furthermore, EGFP transcript and fluorescence levels were significantly induced following HU-treatment.
This system is readily utilizable for genome scale studies of cis-acting regulatory elements which are implicated in γ-globin expression or HU-mediated induction.
KeywordsCRISPR/Cas9 Hydroxyurea γ-Globin induction Knock-in
This work was supported by a grant from the Genetics Research Center, University of Social Welfare and Rehabilitation Sciences (Grant No. 64032).
Table S1—PCR primer sequences used for generating the donor plasmid are given.
Table S2—The top strand oligonucleotide sequences used for cloning the sgRNA templates into the PX459 plasmid are given.
Fig. S1—A comparison of the endogenous γ-globin 5’-UTR sequence with that of EGFP in the donor plasmid. The 10 nucleotide addition shown in the donor plasmid stems from the presence of an EcoRI site (red), generated upon construct assembly, and retention of the EGFP Kozak sequence (CCACCATGG).
Fig. S2—Verification the EGFP cassette knock-in. (a) Targeted integration of the EGFP-NeoR cassette was verified by PCR amplification of the insertion junctions and Sanger sequencing of the ensuing PCR products. Results for the RHA junction are shown. (b) Copy number of the integrated EGFP cassettes was determined by relative qPCR (IC=internal control). The positions of the deployed EGFP and γ-globin PCR primers relative to the DNA ds-break site are shown.
Fig. S3—Analysis of the γ-globin replacement upon EGFP knock-in. (a) Schematic of the possible recombination events between the EGFP knock-in construct and the γ-globin genes (Gγ and Aγ) is shown. (b) Gap-PCR (GFP to Aγ) or PCR (GFP to Gγ) was used to distinguish between the possible recombination outcomes shown in Fig. S3a. The expected amplicon size for either reaction was 1.4 Kb. PCR amplification of the correct region (γ-globin locus) was verified by Sanger sequencing of the amplicon population (data not shown).
Fig. S4—Investigating the presence of indels and deletions in the non-targeted γ-globin copies (a) The γ-globin RNA levels showed a 2-fold reduction per copy in the knock-in cells relative to K562 cells (n=3). (b) This could have been due to presence of indels in the non-targeted γ-globin 5’-UTRs, which was examined by heteroduplex/PAGE analysis. (c) Alternatively, this could have been due to a 4.9 Kb γ-globin deletion, which was investigated by gap-PCR. A 1.4 Kb amplicon was expected for the PCR reactions. Multiple PCR products were observed in the LHA to Gγ reaction, presumably due to the formation of large deletions at the break junctions in the non-targeted Gγ copies.
Fig. S5—Presence of an extra amplicon upon PCR of the LHA in clone-4. (a) The LHAs were PCR amplified from the cellular clones and run on a 1% agarose gel. The forward PCR primer was located outside of the arm, and the reverse primer was located in the transgene. (b) Sanger sequencing of the EGFP start codon region from the amplicons obtained from clones 2 and 4 is shown.
- Banan M, Esmaeilzadeh-Ghar E, Nezami M, Deilami Z, Farashi S, Philipsen S, Esteghamat F, Pourfarzad F, Ali Imam AM, Najmabadi H (2012b) cAMP response element-binding protein 1 is required for hydroxyurea-mediated induction of γ-globin expression in K562 cells. Clin Exp Pharmacol Physiol 39:510–517CrossRefGoogle Scholar
- Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y, Lin C, Shao Z, Canver MC, Smith EC, Pinello L, Sabo PJ, Vierstra J, Voit RA, Yuan G-C, Porteus MH, Stamatoyannopoulos JA, Lettre G, Orkin SH (2013) An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 342:253–257CrossRefGoogle Scholar
- Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, Green RD, Dekker J (2006) Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements. Genome Res 16:1299–1309CrossRefGoogle Scholar
- Naumann S, Reutzel D, Speicher M, Decker HJ (2001) Complete karyotype characterization of the K562 cell line by combined application of G-banding, multiplex-fluorescence in situ hybridization, fluorescence in situ hybridization, and comparative genomic hybridization. Leuk Res 25:313–322CrossRefGoogle Scholar