2018 Fall Webinar Series 1 Webinar Register Strategies to Efficiently Generate CRISPR KO/KI Cell Lines 11:00-12:00PM EST, Oct. 10th Speaker: Shawn Zhou, Ph.D., Senior Scientist, GenScript USA Inc. Register Now >> Bispecific Single-Domain Antibody fused to Monoclonal Antibody (SMAB): The Natural Form 11:00-12:00PM EST, Oct. 17th Speaker: Li Chen, Ph.D., Senior Scientist, GenScript USA Inc.

Register Now >> 3 Tips to Generate The Perfect Immunofluorescent Image 11:00-12:00PM EST, Oct. 24th Speaker: Travis Thomson Ph.D., Assistant Professor, Univ. of Massachusetts Medical School

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2 How to Effectively Use CRISPR in Cell Line Engineering Optimized Workflow and Tips

Presenter： Shawn Zhou, Ph.D. Senior Scientist, GenScript USA Inc.

Date ：Oct 10th, 2018 CONTENTS

CRISPR Background

CRISPR Workflows

Troubleshooting and Tips What is CRISPR/Cas9?

•Ingredients: •Guide RNA(gRNA) •Cas9 protein

Cleavage site: 3-4 bp up to PAM (NGG) (Ding et al., 2016)

5 CRISPR/Cas9 mediated genome editing

• gRNA/Cas9 can be delivered through plasmid-based transfection, virus-based infection, and ribonucleoprotein (RNP)- based microinjection.

• DSBs can be repaired in 2 pathways: • NHEJ pathway (no donor DNA) • HDR pathway

knockout Knock-in

(van Erp et al., 2015)

6 Applications with CRISPR/Cas9 technology

•CRISPR library screening • Gene therapy •Target validation • Cell therapy (CAR-T) •Cell model •Animal model Human Drug therapeutics discovery

Agriculture Industrial bioproduction

• Plant breeding •Bioprocess cell line/strain

7 CAR-T Cell therapy with CRISPR technologies is on trend

(Jung & Lee, 2018. Molecules and Cells) 1. Development of off-the-shelf CAR-T cells (e.g TCR/CD3, B2M knockout) 2. Knockout antigen gene in T cells to prevent self-killing effect (e.g endogenous CS1 knockout in CAR-T) 3. Enhancing anti-tumor activity of CAR-T therapy (e.g knockout immune checkpoint inhibitors, PD1, LAG3 et al.)

8 Clinical trials of cell therapy with CRISPR technologies

9 Engineering non-viral TCR/CAR-T cells with CRISPR

Replace endogenous TCR with antigen-specific TCR by transfection of RNP (synthetic gRNA + Cas9 protein) and DNA donor

(Roth et al., 2018. Nature)

• Webinar: CRISPR Based T Cell Editing: Large knock-ins in human T cells using non-viral HDR templates • Speaker: Theodore Roth, University of California, San Francisco Date: Nov. 1st, 2018 Time: 11:00-12:00 PM EST

10 CONTENTS

CRISPR Background

CRISPR Workflows

Troubleshooting and Tips Workflows for gene knockout/knock-in cell line engineering

3-4 weeks Timeline: 2 weeks 3-4 weeks 3-5 weeks Host cell gRNA design Cell pool Single cell cloning Genotyping 11-15 weeks in total characterization & synthesis generation

Cas9 gRNA Donor

Checkpoint •Mycoplasma test •gRNA design •Transfection or viral •Limit dilution •Sanger Sequencing •Cell culturing •gRNA delivery system infection •FACS sorting •RFLP •Transfection selection •Enrichment of transfected •RT-PCR/qPCR optimization •gRNA/Cas9 synthesis cells by FACS sorting or •Western blot antibiotic selection •Clonality optimization •Functional assay •Promoter validation •Drug selection •HDR efficiency •gRNA cleavage optimization examination •CNV investigation •Gene correction/insertion examination

12 Host cell line characterization

1. Mycoplasma test The host cell line for CRISPR editing must be mycoplasma free (eg. PCR detection kit or Hoechst DNA staining). 2. Cell culturing Select an appropriate host cell line and adjust the cells to good condition before transfection of gRNA/Cas9. 3. Optimization of transfection Test the host cells with different transfection reagent/method. Select an appropriate delivery method (plasmid transfection/lentivirus infection/RNP transfection). 4. Promoter activity test For plasmid/lentivirus-based method, gRNA and Cas9 expression should be driven with strong promoter in the host cells. 5. Clonality optimization • Test and select an appropriate method (Limit dilution or FACS sorting) for single cloning. • Using conditioned medium or single cloning enhancing reagents (e.g Stem cell cloning medium). 6. Gene copy number variation (CNV) investigation • Cancer cell line often carry CNV and the gene may not be diploidy. • CNV investigation is recommended (COSMIC database or ddPCR).

13 gRNA/Cas9 delivery system selection

Plasmid Lentivirus RNP (sgRNA+Cas9 protein) Cost Low Medium High Timeline 11-15 weeks 15-19 weeks 11-15 weeks Cancer, primary and stem Cancer, primary and stem Cell types Cancer cell lines (iPSC) cells (iPSC) cells Any species with appropriate Species Mammalia cells Any species promoters Toxicity Medium High Low Need promoter validation? Yes Yes No Cleavage efficiency Low Medium High Off-target effect Medium High Low Cas9 integration Low efficiency High efficiency No integration Transfection Lipo, electroporation, injection Lentivirus infection Lipo, electroporation, injection Application KO/KI cell line engineering KO cell line engineering KO/KI cell line engineering Best for KO/KI in hard-to- Best for KO/KI in easy-to- Best for KO in hard-to-transfect Summary transfect cancer, primary and transfect cancer cells cancer cells stem (iPSC) cells.

14 Knockout/Knock-in cell pool examination

TIDE/TIDER analysis T7E1/Endonuclease Amplicon-Seq FACS qPCR

Brinkman, et al., 2014

TIDE/TIDER T7E1/RFLP Amplicon-Seq FACS qPCR T7E1 cleaves gRNA- FACS detect KI insertion mediated DNA mismatch Deep sequencing of PCR Deconvolution of Sanger efficiency for fluorescent qPCR probes bind DNA at Mechanism (KO) or endonuclease products for both KO and KI sequencing data marker or target protein by specific site. cleaves HDR-mediated (point mutation) antibody nucleotide replacement Cost Low Low High Medium (need instrument) High Timeline 2-3 days 1 day 1-2 weeks 1-3 hours 1-2 days Sensitivity Medium Low High Medium High Application INDEL, point mutation INDEL, point mutation, INDEL, point mutation insertion INDEL, point mutation, insertion insertion

15 Case study: CRISPR KO with RNP delivery system in THP-1 cells

DNA transfection is very toxic and very hard in suspension cells (e.g THP-1, T cells). RNP transfection presented low toxicity and high cleavage efficiency. HPRT KO in THP-1 cells 1 2

1. Transfected with gRNA/Cas9 2. Parental THP-1 cells Cleavage efficiency: 50%

T7E1 digestion

GenScript RNP Services: https://www.genscript.com/crispr-cas9-protein-crRNA.html

16 Case study: CRISPR KI point mutation with RNP delivery system in U937 cells Procedure 1. Asymmetric ssODN donor with 127 nt is used

gRNA/donor mutation HindIII 2. Silent mutation on homology arm is made to design introduce HindIII recognition site 3. gRNA/Cas9 protein and ssODN are electroporated to U937 cells. 4. PCR product of cell pool is digested with HindIII for KI efficiency evaluation 5. PCR product of clones is digested with HindIII for screening of KI positive clones Cell pool KI efficiency =12.8% examination Design of asymmetric ssODN 1. The Cas9 cleavage site (4th nucleotide before PAM) with 36 bp on the PAM-distal side and with a 91-bp HindIII digestion extension on the PAM-proximal side of the break (including the PAM) 2. Complementary to the gRNA non-target strand (i.e. complementary to gRNA sequence) Clone screening 3. Two nucleotides at either 5’ or 3’ termini are modified with Phosphothioate Oligonucleotide (PTO) (Richardson et al., 2015. NATURE BIOTECHNOLOGY)

17 Case study: CRISPR KI GFP insertion with RNP delivery system in HEK293T cells Procedure gRNA gRNA/donor 1. Long dsDNA and long ssDNA donor are used. 300bp 300bp design 2. gRNA/Cas9 protein and donor are transfected to cells with lipo2000. Fusing GFP to the N-terminal of Rab11a 3. GFP insertion is monitored by FACS

dsDNA ssDNA FACS for GFP fluorescence HDR efficiency Higher Lower Off-target efficiency Higher Lower Cost Lower Higher Toxicity Higher Lower Cell pool 45% of GFP 32% of GFP positive clones positive clones Recommend Easy cancer cell Suspension, primary, examination line (e.g HEK293T) stem, iPS cells GenScript service Gene synthesis Single stranded DNA synthesis

dsDNA donor ssDNA donor GenScript provides ssDNA synthesis service Email us: [email protected]

18 How to effectively generate CRISPR KO/KI Cell Lines?

 Choose the best gRNA/Cas9 delivery method for your host line Using a best transfection method (Lipo, electroporation, lentivirus) Using a best gRNA/Cas9 form (Plasmid, lentivirus, RNP)  Choose the best gRNAs for your target gene Using validated gRNAs  Enrich the transfected cells FACS sorting Antibiotics selection  Increasing HDR efficiency in your host cells Using asymmetric ssODN for point mutation Using long dsDNA donor for gene insertion

19 CONTENTS

CRISPR Background

CRISPR Workflows

Troubleshooting and Tips Issue 1: Low gRNA cleavage efficiency is detected in transfected cells Possible Reasons and Solutions: 1. Low transfection efficiency • Optimize transfection efficiency with a positive control gRNA/Cas9-GFP plasmid/lentivirus/RNP and enrich the transfected cells by FACS sorter or antibiotic selection 2. Low gRNA cleavage activity • Design multiple gRNAs and target different exons • Check gRNA sequence whether PAM is contained in plasmid or synthetic oligos • Using single-stranded RNAs for RNP • Using both N- and C- terminal tagged-NLS Cas9 plasmid or protein (PX458, PX459, et al.) 3. Cell death or growth inhibition after gene knockout • Investigate phenotypes of gene knockout/knockdown through papers before experiments • If cell death or severe growth inhibition, inducible knockout/knockdown is recommended • If KO slow cell growth rate, detect gRNA cleavage efficiency at 24h, 48h, 72h and 96h after transfection and select an appropriate time point to plate single clones

21 Issue 2: Low HDR efficiency is detected in transfected cells

Possible Reasons and Solutions: 1. Low gRNA cleavage efficiency • Refer to the troubleshooting of low gRNA cleavage 2. Sequence difference between genome and homolog arms at donor • Sequencing homolog arms from genome DNA before synthesizing donor 3. HDR pathway activity is low in parental cells • Using an alternative host cell line that has been used for HDR in published paper • Increasing HDR activity by adding drugs (NU7441, NOC, SCR7). Test the drugs effect before selection. • Introduce silent mutation on donor to avoid gRNA re-cutting • Using gRNAs that are close to mutation site (<15bp) • For insertion, using PCR-amplified or restriction endonuclease-linearized double-stranded DNA with long homolog arms (Tild-donor) (Yao et al., 2018 ) • For point mutation, using asymmetric ssODN donor (Richardson et al., 2015)

22 Issue 3: No expected function is observed in KO/KI cells

Possible Reasons and Solutions: 1. Truncated protein still works to maintain the function of the original protein • Using gRNAs that have been validated to disrupt function of the protein in published paper • Design gRNAs to target the area that is homologous to transgenic mouse model (e.g Jacksonlab). • Design gRNAs to target upstream of the functional domain. 2. Alternative spliced isoforms works to supplement function of the original protein • Using double-gRNA strategy to delete the gene from genome 3. Homologous or multiple-copy gene supplements function of the protein • Investigate homologous or multiple-copy genes in host cells by bioinformatics analysis or whole-genome sequencing (e.g COSMIC) • Design gRNAs to target homologous genes 4. Endogenous gene expression level is very low • Investigate gene expression level before selection of the host cell line • Using drugs to stimulate expression of the gene

23 GenCRISPRTM cell line service advantages GenScript generates fully-validated gene knock-out/knock-in cell lines and cell pools using CRISPR technology.

ONE-STOP SOLUTION TRACK RECORD FAST DELIVERY From Design Hundreds of Stable 11-20 Weeks to Functional Assays Cell Lines Delivered for Standard Cell Lines

 Versatile gRNA-Cas9 Vector System, RNP System Dual or AIO vectors carrying various selection markers and Cas9 expressing cassette driven by various promoters  Expertise with Gene Transfection Methodologies Nucleofection (Nucleofector), Electroporation (Celetrix), Chemical, Lentivirus  Strong Customer Support Ph.D level customer representatives available 24/7 to help make your research easy; A dedicated project manager will provide periodic updates throughout the process

24 GenScript CRISPR Reagents and Resources

gRNA/Cas9 Plasmids • Broad Institute pre-validated gRNA and plasmid design • Custom all-in-one or dual eSpCas9, SpCas9, Nickase, and SaCas9 vectors for sgRNA expression CRISPR RNP User Manual • SAM constructs for transcription activation A guide on how to use CRISPR RNP for targeted genome editing. Synthetic gRNAs & Cas9 Nucleases • Fully customizable synthetic sgRNAs, unmodified or modified • Synthetic custom crRNAs and tracrRNAs • Synthetic ssDNA up to 8000nt long in length • SpCas9 Nuclease 2NLS for optimized nuclear compartmentalization • NLS-Cas9-EGFP Nuclease enabling fluorescence activated cell CRISPR Handbook A concise guide to help jump- sorting (FACS) start your gene editing research. Learn more @ https://www.genscript.com/CRISPR-Cas9-technology-resource.html

25 Upcoming Webinar Topic: CRISPR Based T Cell Editing: Large knock-ins in human T cells using non-viral HDR templates Speaker: Theodore Roth, University of California, San Francisco Date: Nov. 1st, 2018 Time: 11:00-12:00 PM EST Questions? Email [email protected] or [email protected]