CRISPR: A Simple Tool for Answering Complex Questions

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1 CRISPR: A Simple Tool for Answering Complex Questions c_bass@bcm.edu

2 CRISPR-Cas systems CRISPRs: Clustered Regularly Interspaced Short Palindromic Repeats Cas: The accompanying CRISPR-associated (Cas) proteins From the Doudna Lab Small crrnas transcribed from CRISPR loci & Cas proteins Prokaryotic adaptive immune response, sequence-specific detection and silencing of foreign nucleic acids. Via base pairing with the crrna guide sequence, leading to Cas protein-mediated DNA cleavage. Three main types (I, II, and III).

3 Type II CRISPR-Cas9 System (SpCas9) PAM Guide RNA

4 Applications of CRISPR-Cas9 Knockout (KO): indels, deletions Knock in (KI): point mutations, epitope tags, reporter sequences CRISPRi: transcription repression (inactive Cas9 fused to transcription repression domain) CRISPRa: transcription activation (inactive Cas9 fused to transcription activation domain) X *

5 Using CRISPR-Cas9 for Genome Editing NHEJ Indels Knockout (KO)

6 Using CRISPR-Cas9 for Genome Editing HDR HDR Knock in (KI)

7 Practical Considerations RNAi (shrna/sirna) CRISPR/Cas9 Target Transcript Genomic DNA Loss of function Reversible KD Irreversible KO Type of phenotype hypomorphic Null Time Short Long Ease Easy More difficult Cost Low High Off-targeting High Low*

8 Generating CRISPR KO/KI Cell Lines Cells of interest. Gene of interest Cas9 sgrna (crrna + tracrrna) Plasmid(s) RNA/mRNA RNP All-in-one Split Inducible Viral Donor template (KI) (ssodn/fragment) Delivery Transfection (plasmids, RNA, protein/rna) Injection (DNA, RNA) Viral transduction

9 Generating CRISPR KO/KI Cell Lines Cell cloning Cas9, grna, (donor) To obtain monoclonal cell populations Limiting dilution Single-cell sorting Clone analysis Isolate and expand individual clones Confirm KO/KI PCR Sequencing Western blotting Immunostaining ELISA

10 Scientific Examples (1) Using a CRISPR KI breast cancer cell line to study estrogen receptor (ER) mutations and metastatic breast cancer U6 ssodn grna Cas9 egfp Guowei Gu and Suzanne Fuqua (2) Using inducible CRISPR KO cancer cell lines to study telomere dysfunction Tet-on U6 Cas9 grna Hyeung Kim and Zhou Songyang

11 Estrogen Receptor (ER) Mediates Gene Transcription ON- Estrogen Dependent OFF ON- Estrogen Independent ER ER ERm Zhang 1997 E 2 E 2 Gene Gene Transcription Transcription? ERm Metastatic Primary Tumor Tumor Growth Growth? Resistance? Response E 2 San Antonio Breast Cancer Symposium December 6-10, 2016 This presentation is the intellectual property of the author/presenter. Contact sfuqua@bcm.edu for permission to reprint and/or distribute

12 This presentation is the intellectual property of the author/presenter. Contact me at for permission to reprint and/or distribute San Antonio Breast Cancer Symposium December 6-10, 2016 Mutations in the Gene Encoding ERα (ESR1) and Metastatic Breast Cancer (MBC) 20-40% of MBC contain ESR1 gene mutations, acquired during treatment of MBC with aromatase inhibitors (AI) An AI alone may be countraindicated in a MBC patient with ESR1 mutations Fulvestrant is effective in MBC with ESR1 mutations herα

13 San Antonio Breast Cancer Symposium December 6-10, 2016 Y537S ESR1 Mutation Model ESR1 * exon 8 Cas9, grna-2, ssodn donor T7 assay for Cas9 cleavage efficiency grnas MW MCF-7 Genomic DNA sequencing * Cells homozygous knocked in for the Y537S mutation (YS1 and YS30) This presentation is the intellectual property of the author/presenter. Contact me at sfuqua@bcm.edu for permission to reprint and/or distribute

14 This presentation is the intellectual property of the author/presenter. Contact me at for permission to reprint and/or distribute San Antonio Breast Cancer Symposium December 6-10, 2016 Y537S ESR1 Mutation Model Estrogen Regulated Genes Estrogen Independent Transcription P/WT YS1 YS30 ER PR c-myc CCDN1 GAPDH R eporter A ctivity (L u c/ β - g a l) Parental ** Y S 1 ** Y S 3 0 Guowei Gu -E2 Pronounced Tamoxifen Resistance Responsive to Fulvestrant Growth Assay (Fold) P W Y S 1 T Growth Assay (Fold) P W Y S 1 T C 1 n M 1 0 n M n M 1 u M 1 0 u M Tamoxifen C 1 n M 1 0 n M n M 1 u M 1 0 u M Fulvestrant

15 This presentation is the intellectual property of the author/presenter. Contact me at for permission to reprint and/or distribute San Antonio Breast Cancer Symposium December 6-10, 2016 Y537S ESR1m Induces an Epithelial to Mesenchymal Transition (EMT) mesenchymal fibroblastic Lose contact inhibition and cell polarity e.g., enhanced migratory capacity, invasiveness, and resistance to apoptosis. EMT a mechanism for the initiation of invasive and metastatic epithelial cancers

16 This presentation is the intellectual property of the author/presenter. Contact me at for permission to reprint and/or distribute San Antonio Breast Cancer Symposium December 6-10, 2016 Cell Mixing Experiment Mixing WT + YS1(M) WT M/WT 1% 10% 50% 90% E2 E2 E2 E2 Ovariectomized nude mice Primary tumor growth Metastases (macro-met and micro-met)

17 San Antonio Breast Cancer Symposium December 6-10, 2016 The Y537S ESR1 Mutant Has Competitive Proliferative Advantage and Drives Metastasis Mutant Frequency (ddpcr) % 10% Mutant 1 % P r im a r y 1 % M e t 1 0 % P r i m a r y 1 0 % M e t 5 0 % Prim ary 5 0 % M e t 1% 10% 50% 100% 9 0 % P r im a r y 9 0 % M e t 50% 90% M a c r o M e t a s t a s i s F r e q u e n c y Macro Metastasis Frequency % Macro-Met 1 0 % M e ts * 8 0 * 5 0 % 9 0 % This presentation is the intellectual property of the author/presenter. Contact me at sfuqua@bcm.edu for permission to reprint and/or distribute L u n g M e t a s t a s i s F r e q u e n c y Lung Micrometastasis frequency % Lung Micro-Met 1 0 % 5 0 % 9 0 % M e ts

18 This presentation is the intellectual property of the author/presenter. Contact me at for permission to reprint and/or distribute San Antonio Breast Cancer Symposium December 6-10, 2016 ESR1 Mutation Summary and Clinical Implications Mutations are a bona-fide mechanism of acquired hormone resistance First preclinical evidence that ESR1 mutations also enhance tumor progression and metastatic behavior

19 Telomeres are repetitive sequences at the ends of linear chromosomes buffers. (TTAGGG) Telomere shortening is associated with aging. Telomere dysfunction has been linked to agingrelated diseases and cancer.

20 The Telosome Maintains Telomere length and Integrity Telomere loss DNA damage Telosome/Shelterin Hyeung Kim

21 Generating Inducible CRISPR KO Cells to Study Telomere Regulation by the Telosome TRE Inducible Cas9 (lentiviral) Drug Selection -dox HeLa cells expressing inducible Cas9

22 Generating Inducible CRISPR KO Cells to Study Telomere Regulation by the Telosome Inducible Cas9 (lentiviral) -dox Drug Selection grna(s) TIN2 TRF2 TRF1 TPP1 RAP1 POT1 -dox -dox -dox -dox -dox -dox Drug Selection ±dox ±dox ±dox ±dox ±dox ±dox

23 Generating Inducible CRISPR KO Cells for All Six Telosome Subunits

24 Dual grnas Lead to More Complete KO Cas9

25 Inducible CRISPR KO Cell Lines for All Six Telosome Subunits

26 Using Inducible CRISPR KO Cells to Study Essential Genes

27 KO Individual Telosome Subunits Disrupts Telosome Targeting to Telomeres Telomere ChIP experiments Individual telosome subunits were IPed +/-doxycycline The amount of co-precipitated telomere DNA was quantified

28 KO Individual Telosome Subunits Disrupts Telosome Targeting to Telomeres (cont d) Low ChIP ratios, reduced binding to telomeres

29 KO Individual Telosome Subunits Disrupts Telomere Maintenance Telomere fusion Normal Telomere loss TRF1-DG (-Dox) TRF1-DG (+Dox) TRF2-DG (-Dox) TRF2-DG (+Dox) TIN2-DG (-Dox) TIN2-DG (+Dox) TPP1-DG (-Dox) TPP1-DG (+Dox) POT1-DG (-Dox) POT1-DG (+Dox) RAP1-DG (-Dox) RAP1-DG (+Dox) Telomere fusions Telomere loss 0 0.7% 0 7.1% 0.1% 1.2% 73.1% - 0.3% 0.9% 8.3% 19.6% 0 1.1% 0 3.5% 0 2.7% 0 7.8% 0 1.1% 0 9.6%

30 Generating CRISPR KO/KI Cell Lines Cells of interest. Gene of interest. Cas9 sgrna (crrna + tracrrna) Plasmid(s) RNA/mRNA RNP All-in-one Split Inducible Viral Donor template (KI) (ssodn/fragment) Delivery Transfection (plasmids, RNA, protein/rna) Injection (DNA, RNA) Viral transduction

31 C-BASS Acknowledgement Collaborators Trey Westbrook Guowei Gu Suzanne Fuqua Jun Xu Hyeung Kim Zhou Songyang