Cell Culture Techniques Lecture 7+8: Genetic Modification, RNAi, & CRISPR-Cas Applications PDF

Cell Culture Techniques Lecture 7+8 Genetic Modification RNAi CRISPR/Cas applications Andreas Lackner Fri, 08.11.2024 Bachelor Program Medical and Pharmaceutical Biotechnology  The entire lecture at a glance...

Cell Culture Techniques Lecture 7+8 Genetic Modification RNAi CRISPR/Cas applications Andreas Lackner Fri, 08.11.2024 Bachelor Program Medical and Pharmaceutical Biotechnology  The entire lecture at a glance Cycle normal Microscopy Senescence Light, Dyes, Antibodies FACS aberrant Metabolic Proliferation Chemicals, Toxicity Antibodies, Cancer Cell Based Assays Cells Differentiation Cell death Diseases Genetic modification Death KD OX KO Reporter/Tag Apoptosis CRISPR/Cas9, RNAi, Transduction Transfection, Plasmids Necrosis How to evaluate gene/protein function Genomic editing Overexpression Reporter Assays RNAi (siRNA/shRNA) History of RNAi  Knock down of a Gene of Interest RNA interference (RNAi) is using an endogenous machinery The tools are available! Never forget side effects by overloading the endogenous machinery  Knock down of a Gene of Interest RNA interference (RNAi) is using an endogenous machinery RNAi employs miRNA machinery https://www.cell.com/molecular-therapy-family/nucleic- acids/fulltext/S2162-2531%2823%2900191-9  What happens during RNAi ? RNAi is a biological process siRNA levels Target the mRNA/lncRNA of Endogenous mammalian a gene-of-interest microRNA mechanism Reduce the level of involved mRNA levels protein/lncRNA mRNA v. Protein levels not directly proportional Protein levels Unintended You produced more consequences are the Δ in related biological questions than answers rule, not the exception pathway(s) You are on the right way! Time point, assay used, Δ in cellular biology cell type, etc. can lead to Something wasn’t as different results expected Δ in phenotype Never expect anything!  Knock down of a Gene of Interest How can a gene/protein be down regulated/knocked down? − Inhibit function eg. small molecule inhibitors − Inhibit expression by interfering with mRNA − Induce protein degradation (degron, PROTAC) Oligo based- siRNA Plasmid based- shRNA transfection Usually transduced Small interference RNA Components Small hairpin RNA double-stranded RNA to be processed in the cell 20-27 bp in length into a siRNA oligo + easier to transfect versus - Harder to transfect + fast experiments - Long term experiment - only transient effect + stable effect Oligo or plasmid based? Depends on cell model Depends on mRNA/Protein Depends on research question Applications of RNAi Applications of RNAi Where does CRISPR/Cas9 work? Genomic editing/interference RNA editing/interference Reporter Assays What's this 'CRISPR', everybody is talking about? Did humans invent it?  Theoretical Background- Mechanism CRISPR: Clustered Regularly Interspersed Short Palindromic Repeats Cas: CRISPR-associated proteins, a nuclease PAM: Protospacer adjacent Motif Mechanism of adaptive immunity in bacteria and archaea Evolved to adapt and defend against foreign genetic material (i.e., phage, horizontal gene transfer, etc.) Used now in eucaryotic cells as tool to modify the genome Bhaya et al., Annu. Rev. Genet. 45, 273-97 (2011) History of Genome Editing Tools J Doudna& E Charpentier Genome editing. The new frontier of genome engineering with CRISPR-Cas9 (2014)  Theoretical Background- Mechanisms of DNA repair Editing by repair of double-strand breaks (DSB) nuclease–induced DSB Non-homologous end joining Homology-directed repair (HDR) (NHEJ) knockouts mutations, knockins, fusions donor DNA template deletions insertions (indels) variable lengths precise insertion or modification JD Sander & JK Joung, Nature Biotechnology (2014) History of Genome Editing Tools J Doudna& E Charpentier Genome editing. The new frontier of genome engineering with CRISPR-Cas9 (2014) The CRISPR-Hype started in 2014… Forscher verspeiste erstmals Crispr-Pflanze Der Standard, 9. September 2016 Emanuelle Charpentier (till 2009 at University of Vienna)  History of CRISPR gene therapy https://www.frontiersin.org/articles/10.3389/fonc.2020.01387/full  CRISPR/Cas9 in the lab Tracer RNA Guide/crRNA Cas Nuclease Genomic DNA single guide RNA: Fusion of the programable part (gRNA or crRNA) and the Tracer RNA  Theoretical Background- Mechanism Cas9 is bacterial – Do we have to change anything to make it work in eurkaryotic cells? Nuclear localization sequence What do you need to edit a genome? Cas9…Nuclease taken from J Doudna& E Charpentier, 2014, DOI: 10.1126/science.1258096 CRISPR/Cas subtypes  CRISPR/Cas subtypes Interesting? RNA editing? Is there a practical implication?  CRISPR/Cas13 and anti-viral defense The way from first basic research hypothesis to therapy is far! 5-20 years https://doi.org/10.1016/j.cell.2020.04.020  CRISPR/Cas subtypes Summary spCas9 saCas9 Cas12 Cas13 Protein size 163kDa 130kDa 152kDa 130kDa Target dsDNA dsDNA dsDNA ssRNA PAM Seq 3‘NGG 3‘NNGRRT 5‘TTTN 3‘H or 5‘D guideRNA 18-24 nt 18-24 nt 42-44nt 22-30nt length Cut Blunt Blunt 5‘ overhang of nonspecifc 5nt origin Streptococcus Staphylococcus Francicella Listeria pyogenes aureus novicida seeligeri  CRISPR/Cas9 binding mechanism Protospacer adjacent motif (PAM) is necessary for Cas9 residence on DNA increasing complementarity of the guide increases residence time nuclease activity at high residence times Nature 507(7490), 62–67 doi:10.1038/nature13011 Mechanisms of DNA repair and genome editing Editing by repair of double-strand breaks (DSB) nuclease–induced DSB Non-homologous end joining Homology-directed repair (HDR) (NHEJ) knockouts mutations, knockins, fusions donor DNA template deletions insertions (indels) variable lengths precise insertion or modification JD Sander & JK Joung, Nature Biotechnology (2014) Tools and work flows for CRISPR/Cas9 experiments in cell culture What do you need (to know)? How do you apply it?  Which tools are available? Almost all ! AAV Cas9 and sgRNA Adenoviral Cas9 Viral lentiviral Cas9 and sgRNA Plasmid-based All-in-one plasmid Cas9+sgRNA Cas9 plasmid Cas9 Non-Viral +/- fluorophore expressing cells lines Mixed version sgRNA plasmid Donor construct mRNA Cas9 Plasmid-free Cas9 proteins crRNA oligos/libraries DNA template  Strategies for gene Knock Out (KO) – How to kill a protein We want to generate a truncated or completely deleted protein by: 1.) Creating a random „indel“ (insertion or deletion), which causes a premature STOP codon Difficult to analyze (Sanger sequencing) In-frame possible Truncation possible 2.) Deleting one or more exons (spanning conserved domains) Easier to analyze (PCR) In-frame possible Potential of splicing escapees Truncated protein possible 3.) Creating an „exon-intron“-fusion Easy to analyze (PCR) In-frame unlikely Definitive degradation  What features does a gene have? Exons: the coding part of a gene Introns: non-coding, but important regulatory features (transcription, splicing) UTRs: regulatory regions (translation) https://en.wikipedia.org/wiki/File:Gene_structure_eukaryote_2_annotated.svg Strategies for gene Knock Out (KO) - Creating a random „indel“ One guide RNA (1 exon)  Strategies for gene KO – Deleting one or more exons Two guide RNAs (2 exons) deletion deletion  Strategies for gene KO – Creating an „exon-intron“-fusion Two guide RNAs (1 exon, 1 intron) deletion deletion Exon intron fusion – additional KO chances  Strategies for gene Knock Out (KO) – How to kill a protein We want to generate a truncated or completely deleted protein by: 1.) Creating a random „indel“ (insertion or deletion), which causes a premature STOP codon Difficult to analyze (Sanger sequencing) In-frame possible Truncation possible 2.) Deleting one or more exons (spanning conserved domains) Easier to analyze (PCR) In-frame possible Potential of splicing escapees Truncated protein possible 3.) Creating an „exon-intron“-fusion (spanning conserved domains) Easy to analyze (PCR) In-frame unlikely Definitive degradation Timeline of a CRISPR based genetic modification CRISPR stable Cell line Workflow-Knock in or Knock out (3 weeks - 8 months) Transfer of plasmids into the cell Test for other potential defects (off targets, chromosomal aberrations...) Enrich the edited cell population ….because, every single cells has an individual mutation behaviour ….analyze for successful modification  Knock-Out Workflow- Time Line Expert level / Students level 1. ‚Design‘/Select sgRNAs for your KO (chopchop) – 1h / up to 1 week 2. Order oligos for sgRNA cloning – 2-3 days / 2-3 days 3. Clone sgRNAs into All-in-one vector – 3-4 days / 2-3 weeks 4. Transfer Cas9/sgRNA plasmid to the cells – 1 day / 2-3 attempts 5. Select for transfectants – 2 days / 2-3 attempts 6. Seed cells for single cell cloning – 1h / 2-3 h 7. Grow to „pickable“ colonies (depending on cell line) – 6-20 days 8. Analyze for successful knock-out – 1-2 days / 1 week ES/TS cells (mouse): 16-20 days total / 10 weeks ES/TS cells (human): 20-30 days total / 3 months ES...embryonic stem cells TS...trophoblast stem cells Knock-Out Workflow – sgRNA cloning Where do you amplify the plasmid? Knock-Out Workflow – sgRNA cloning sgRNA seq U6 promoter (Pol III/small RNAs) sgRNA sequence Mammalian (!) Promoter Cas9 Mammalian (!) antibiotic selection marker All-In-One Vector sgRNA/Cas9 Which parts do you change for conferring specificity for your gene of interest?  Knock-Out Workflow- Time Line Expert level / Students level 1. ‚Design‘/Select sgRNAs for your KO (chopchop) – 1h / up to 1 week 2. Order oligos for sgRNA cloning – 2-3 days / 2-3 days 3. Clone sgRNAs into All-in-one vector – 3-4 days / 2-3 weeks 4. Transfer Cas9/sgRNA plasmid to the cells – 1 day / 2-3 attempts 5. Select for transfectants – 2 days / 2-3 attempts 6. Seed cells for single cell cloning – 1h / 2-3 h 7. Grow to „pickable“ colonies (depending on cell line) – 6-20 days 8. Analyze for successful knock-out – 1 day – 1 week / up to 2 weeks ES/TS cells (mouse): 16-20 days total / 70 days/10 weeks ES/TS cells (human): 20-30 days total / 90 days/3 months ES...embryonic stem cells TS...trophoblast stem cells Seed cells at single cell dilution or separate wells PCR Proliferation assay Apoptosis assays qPCR Western Blot Cell Culture Techniques 10 min  Strategies for gene Knock In (KI) – How to modify a protein We want to generate a modified protein by: 1.) Creating a site directed mutation (insertion or deletion), causing a functional modification of your protein 2.) C-terminal and N-terminal Knock-In Strategies for gene Knock In (KI) - Creating a directed gene mutation donor oligo Strategies for gene Knock In (KI) - C- and N-terminal knock-in N-terminal: target START C-terminal: target STOP Strategies for gene Knock In (KI) – Donor  Fluorescent Protein Knock In – Fusion vs Bicistronic Potential interference with function Localization/Interaction Bicistronic: Two proteins from one mRNA 2A sites: self cleaving (T2A, P2A, F2A) IRES: 2nd translation start (internal ribosome entry site) Interference with function unlikely No localization/interaction https://en.wikipedia.org/wiki/2A_self-cleaving_peptides#/media/File:2A_peptide_Working_Mechanism.jpg  Knock-In Workflow- Time Line Expert level – Students level 1. Design Donor construct with (micro-)homology arms – 1 week - ? 2. ‚Design‘/Select gRNAs for your KO (chopchop) – 1h - up to 1 week 3. Order oligos for gRNA cloning – 2-3 days to 2-3 days 4. Clone gRNAs into All-in-one vector – 3-4 days to 2-3 weeks 5. Transfer Cas9/sgRNA plasmid to the cells – 1 day to 2-3 attempts 6. Select for transfectants – 2 days to 2-3 attempts 7. Seed cells for single cell cloning – 1h to 2-3 h 8. Grow to „pickable“ colonies (depending on cell line) – 6-20 days 9. Analyze for successful knock-in – 1-2 days to 1 week ES cells (mouse): 16-20 days total to 70 days/10 weeks ES cells (human): 20-30 days total to 90 days/3 months ES...embryonic stem cells  Tags and why to use them Tags are generally fused Smaller than FP → less likely interfering with protein function (unless you want it to – see degron) Can be used for localization/interaction studies (HA, Flag, Myc, etc.) Can be used to generate inducible Kos (Degron- systems: AID, dTag) Donor template for KI can be synthesized!! (Faster/Cheaper than molecular cloning)  Knock-In Workflow 1) Protein is expressed in cell type PCR to detect successful insertion Microscope/Flow cytometer (fluorescent reporter) Immunofluorescence -> Microscope/Flow cytometer (tags) 2) Protein is not expressed Additional steps: induce protein of interest (eg. Differentiate cells, activate pathway of your POI)  Degron systems – dTag system Reversible protein degradation Resembles a reversible/inducible gene KO Tag is fused to the protein of interest Small chemicals used to induce degradation by bridging the introduced tag with an E3 ligase Degron systems – dTag system  Take home message- Design your experiments carefully! not only valid for cell culture techniques What is the scientific question/hypothesis behind? What kind of manipulations are planned? Which cell system is suitable for the planned experiment: cell type, species,... What is the read-out of the experiment mRNA -> RT-qPCR, Microarray... protein -> Western blot, immunofluorescence,... promoter activity -> luciferase assay,... differentiation -> differentiation assay,... morphology -> microscopy,... How to imply statistical significance: how many parallel samples/repeats? What time frame is required for setup and experiment: days, week, months? Which controls have to be included: positive/negative control, mutants,... dCas9 – taking CRISPR beyond DNA editing CRISPRa dCas9 dCas9, nuclease deficient- mutant Equipped with different features by fusion No DNA repair involved (unless the fusion protein is a nuclease) dCas9 – taking CRISPR beyond DNA editing 1) CRISPRa Direct activation (transactivator domains) Indirect activation (epigenetic landscape) 2) CRISPRi Direct repression (transactivator domains) Indirect repression (epigenetic landscape) 3) Epigenome editing Fusion with epigenetic modifier 4) DNA labeling Fusion with NanoLuc (modified Luciferase) dCas9 – taking CRISPR beyond DNA editing https://bitesizebio.com/wp-content/uploads/2020/03/Figure-3.jpg  dCas9 – taking CRISPR beyond DNA editing https://www.researchgate.net/profile/Xiaoshu-Xu- 3/publication/326004058/figure/fig2/AS:647994978357249@1531505450209/CRISPR-dCas9-based-epigenome- engineering-The-epigenetic-landscape-of-DNA-and-histone-can.png DNA looping measurements Screening with CRISPR/Cas9 – pooled screen CRISPR pooled Library Workflow (4-8 weeks) Cancer cell Cytostatic Survivors drug Research Biological question read out Screening with CRISPR/Cas9 – arrayed screen Small CRISPR-Cas9 Screening Library Predesigned for human and mouse − crRNA oligos − Lentiviral sgRNA Whole genome Library Pathways Libraries Apoptosis, Drug Targets, Epigenetic,… Customized defined Format: 96- and 384-well plates  The entire lecture at a glance Cycle normal Microscopy Senescence Light, Dyes, Antibodies FACS aberrant Metabolic Proliferation Chemicals, Toxicity Antibodies, Cancer Cell Based Assays Cells Differentiation Cell death Diseases Genetic modification Death KD OX KO Reporter/Tag Apoptosis CRISPR/Cas9, RNAi, Transduction Transfection, Plasmids Necrosis  Cell Culture Techniques Thanks for your attention! Please, send me the questions per teams_PM Other CRISPR systems in human cells