Controllable Gene KnockoutUsing Piezo1-induced Gene Activation

Haewon Jeong1, Sanup Yang1, Yirye Hong1, Wookjin Shin1, Jung-uk Lee1,3, Chiyo Mikuni1,2,,Chang Ho Sohn1,2* and Jinwoo Cheon1,2,3*

1Center for Nanomedicine, Institute for Basic Science (IBS), Seoul, Republic of Korea2Graduate Program of Nano Biomedical Engineering (NanoBME), Yonsei – IBS Institute, Yonsei University, Seoul, Republic of Korea

3Department of Chemistry, Yonsei University, Seoul, Republic of Korea

Introduction

Result

Conclusion

References

Fig. 1. COSMIC mutation result of KRAS gene mutation of A549 has mutated sequence in 34G>A.

Fig. 3. Single guide RNA (sgRNA) sequence design for gene editing. (A) Mutation site targeting. (sgRNA1-WT, sgRNA2-mutated type) (B) Minimum off-targeting. (sgRNA3) (C) Maximum on-targeting. (sgRNA4)

BA C

Fig. 5. T7-E1 Assay Progress. (A) Scheme of T7-E1 Assay (B) Primer sequence for T7-E1 PCR amplification. (C) Optimization of Rank1 primer Tm for sgRNA1, sgRNA2, sgRNA3. (D) Optimization of the highest efficiency primer Tm for sgRNA4 .

A

Gene Editing

Gene Induction

Fig. 6. Cloning process for generating TetO-mCMV-pGF plasmid. (A) Scheme for cloning. (B, C) Gel electrophoresis result for (B) infusion backbone vector and (C) insert vector after PCR. (D) Colonies for TetO-mCMV-pGF.

Fig. 4. Fluorescence microscopy images of Cas9-RFP overexpressed H1299 cells after transfection with sgRNA-GFP vector. Images of H1299 expressing RFP and GFP were taken 2 days after the sgRNA transfection. (A) Control Group- sgRNA not transfected. (B) sgRNA1 transfected. (C) sgRNA2 transfected. (D) sgRNA3 transfected. (E) sgRNA4 transfected.

D

Fig. 9. Comparison of gene induction level between CSN system and Tetracycline system by counting the GFP-expressing cells. (A, B) Fluorescence microscope images for (A) Tet transfected-Tetracycline treated and (B) CSN transfected-DMSO treated. (C) Induction efficiency of Tet transfected-Tetracycline treated group and CSN transfected-Yoda1 treated group.

B CSN-Yoda1A CSN-DMSO C

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Fig. 2. A549 Cells were transfected with Cas9-RFP plasmid using lipofectamine. (A) Fluorescence microscopy images were taken after the day of transfection. Cells underwent Puromycin selection (1.5 μg/mL), creating a stable cell line. (B) Cas9-RFP overexpressed H1299 stable cell line.

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Fig. 8 Calcium influx analysis using c-Fos. Piezo1+Yoda1- group and Piezo1+Yoda1+ group. (A) RT-PCR was done to compare c-Fos mRNA levels. c-Fos levels increase in the P+/Y+ group compared to the P+/Y- group indicating calcium influx and gene expression.(B) Immunocytochemistry image for c-Fos protein. Nuclei; blue, Piezo1; green, c-Fos; magenta. (C) Average fluorescence intensity of Piezo1 and c-Fos protein in each group.

473 bp -

184 bp -177 bp -

Piezo1: 473 bpβ-actin: 184 bp

c-Fos: 177 bp

P+/Y- P+/Y+ P+/Y- P+/Y+ Piezo + Piezo -Yo

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ΔF % =𝐹𝐹𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 − 𝐹𝐹(−,−)

𝐹𝐹(−,−)× 100 (%)

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two tailed Student’s t-testn.s.; no significant*; P < 0.05**; P < 0.01

Nucleus Piezo1 c-Fos Piezo1 c-Fos

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KRAS is one of the most active oncogenes in human cancers,with 17~25% of human carcinogenic tumors harboring theKRAS mutation(1). It plays a crucial role in initiating andeventually activating the growth-related downstreampathways. Therefore, KRAS mutation or overexpression ofwildtype KRAS may lead to uncontrollable cell proliferation,thereby causing tumor. The KRAS mutation result fromsingle amino acid substitutions in codon 12, 13 or 61 whichleads to the GTPase in a constantly active state by bindingto GTP. In this study we attempted to create a modeldepicting the KRAS oncogene using the wild type H1299cell line and inducing a specific G to A mutation. Then wetarget the KRAS single G to A mutation in the H1299 cellline using the CRISPR-Cas9 gene editing technology.Furthermore, we create an inducible system using themechanosensitive ion channel, Piezo1 to induce the

expression of certain genes. The conversion of mechanical force to chemical signals is essential to biological systems, includingtouch, pain, hearing etc.(3) Piezo1 channels open in response to mechanical stimuli and transmit cationic currents into the cell,which leads to variations in gene expression . We use Yoda1, the chemical activator of Piezo1, to stimulate the ion channel andpromote calcium influx into the cytoplasm to ultimately increase the expression of our target genes GFP and firefly luciferase.Through the activation and deactivation of Piezo1, we introduce an inducible system where the expression of gene editingmolecules such as CRISPR-Cas9 can be controlled. We use the tetracycline operator as a well known positive control(2) andcompare the gene activation efficiencies of Tetracycline and Piezo1.

Fig. 7. Calcium influx analysis using X-Rhod1. Fluorescence intensity increased significantly in Piezo1 overexpressed A549 cells after Yoda1 treatment. (A-D) Real time images of (A) Piezo1-Yoda1- group, (B) Piezo1+Yoda1- group, (C) Piezo1-Yoda1+ group, and (D) Piezo1+Yoda1+ group, respectively. (E) Intensity plot of normalized fluorescence of Piezo1+Yoda1+ group reveals increased fluorescence intensity after Yoda1 treatment. (n=5) (F) Relative Fluorescence intensity indicating huge influx of calcium in Piezo1+ Yoda1+ group (n=5).

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Comparison of GFP expression

In this study, we attempt to confirm the indel mutagenesis of CRISPR-Cas9 and create a controllable gene activation system using the Piezo1 mechano-transduction and subsequent calcium influx. Inducible systems provide key advantages over constitutive gene expression, providing controllable gene activation and regulation of biological processes. However, further study is required to combine the gene editing CRISPR-Cas9 system and Piezo1 to create a system where Piezo1 is stimulated in order to induce the activation of Cas9 and sgRNA. Furthermore, Piezo1 stimulation could also be improved using magnetic nanoparticles instead of the chemical activator, Yoda1. Magnetic fields can be transformed to mechanical stimuli using MNPs and this manipulation of MNPs can be used to target specific tissues and biomolecules.(4)

Utilizing MNPs can be a more biocompatible method to mimic the actual mechano-stimulation in biological systems.(5)

Thus, the Piezo1 mechano-transduction combined with the CRISPR-Cas9 gene editing technology has numerous potential to be used in therapeutic applications.

C 66 65.5 64.5 63 61.2 59.9 58.8 58 oC D 66 65.5 64.7 63.4 61.8 60.6 59.7 59 oC

431 bp -

926 bp -

B Rank 1 Forward 5'- TTCTTAAGCGTCGATGGAGGR-3'

Rank 1 Reverse 5’-GGTTTCTCTGACCATTTTCATGAG-3’

Highest Efficiency Forward 5’- AGGTGCACTGTAATAATCCAGAC-3'

Highest Efficiency Reverse 5’- CCACTGTTTATCCAATCCAAGCA -3’

Expected PCR product: 926 bpExpected PCR product: 431 bp

Kranenburg, Onno. “The KRAS oncogene: past, present, and future.” Biochimica et biophysica acta1756, no. 2 (2005): 81-82.Shockett, Penny, Michael Difilippantonio, Nathan Hellman, and David G. Schatz. “A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice." Proceedings of the National Academy of Sciences 92, no. 14 (1995): 6522-6526.Coste, Bertrand, Jayanti Mathur, Manuela Schmidt, Taryn J. Earley, Sanjeev Ranade, Matt J. Petrus, Adrienne E. Dubin, and Ardem Patapoutian. "Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels." Science 330, no. 6000 (2010): 55-60.Kim, Ji-wook, Hee-kyung Jeong, Kaden M. Southard, Young-wook Jun, and Jinwoo Cheon. "Magnetic Nanotweezers for Interrogating Biological Processes in Space and Time." Accounts of chemical research 51, no. 4 (2018): 839-849.Iskratsch, Thomas, HaguyWolfenson, and Michael P. Sheetz. "Appreciating force and shape—the rise of mechanotransduction in cell biology." Nature Reviews Molecular Cell Biology 15, no. 12 (2014): 825-833.Team:SUSTech Shenzhen/Design, IGEM Team, 20 Oct. 2016, 02:29, 2016.igem.org/Team:SUSTech_Shenzhen/Design.

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