TECHNOLOGIES. 3/19/18 Kayla Nygaard.
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1 TECHNOLOGIES 3/19/18 Kayla Nygaard
2 CRISPR IN THE NEWS CRISPR in 2018: Coming to a Human Near You Sickle-cell treatment clinical trials CRISPR Therapeutics Stanford - MIT Technology Review Doctors in China Lead Race to Treat Cancer by Editing Genes Editing patient T cells - NPR, Feb. 21 st,
3 OUTLINE History of CRISPR/Cas Review of Original CRISPR/Cas9 tech New CRISPR Applications Designing your own CRISPR experiment
4 HISTORY OF CRISPR-CAS Bacterial immune system Targeted ds viral DNA Acquired immunity 1. Injection of ds viral DNA 2. Recognized by Cas Creates novel spacer 1. Transcription of CRISPR array 2. Cas processes CRISPR array 3. crrnas incorporated into CAS complex 4. Inactivation of viral DNA
5 VARIATIONS OF CRISPR 6-8 components 4-5 components 6-8 components 3-4 components 2-4 components 3 components
6 TYPE II: CRISPR-CAS9 CRISPR-Cas9 system Simpler CRISPR system Found in Streptococcus pyogenes 4 components system Cas 1 Cas 2 Cas9 endonuclease tracrrna crrna processed from primary transcript
7 HISTORY OF CRISPR-CAS9 Won Breakthrough of the Year in 2015 Jennifer Doudna UC-Berkeley With Emmanuelle Charpentier Cas9 + synthetic guide RNA! Fuse crrna & tracrrna = grna TED Talk: ow_edit_our_dna_but_let_s_do_it_wisely/up-next
8 ORIGINAL CRISPR APPLICATION How it works: CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats Cas9 = CRISPR-associated endonuclease Cuts DNA grna = guide RNA Targets Cas9 to DNA Applications: Targeted gene editing
9 ORIGINAL CRISPR APPLICATION Targeted gene editing Exploits endogenous repair mechanisms NHEF HDR Optional Activity: 2 person Cas-9 (recognize/cut) Everyone else has original DNA When cut, roll die: 2,4,6 = perfect fix 1,3,5 = NHEJ or HDR Cas9 sit down when you can t cut anymore
10 NEW CRISPR APPLICATIONS CRISPRi CRISPRa CRISPR imaging CRISPRainbow Single base editing Temporal control of CRISPR Multiplexed editing RNA editing Epigenetic modifications enchip
11 CAS9 VARIANTS spcas9 Wild type Cas9 Makes ds breaks in DNA Cas9-HF High fidelity espcas9 Enhanced specificity dcas-foki FokI non-specific nuclease only cuts when dimerized Off targets less likely Cas9n Cas9-nickase single strand break D10A or H840A Enhance specificity SaCas9 dcas9 Staphylococcus aureus spcas9 orthologue different PAM 5 -NNGRRT-3 Mutate both catalytic residues D10A and H840A Unable to cleave dsdna NmCas9 N. meningitidis 5 -NNNNGATT-3
12 CRISPR-INTERFERENCE Developed by Lim & Weissman labs at UCSF in 2013 CRISPR-Interference (CRISPRi) dcas9 = dead Cas9 Regulates at transcription level Sterically: Block initiation or elongation 99.9% repression in prokaryotes 90% repression in human cells Via effector domain: Krüppel associated Box (KRAB) 99% repression in human cells
13 CRISPR-INTERFERENCE ADVANTAGES Repression up to 99.9% Straightforward selection of targetable sites. Multiple sgrnas Multiplexing (more than 1 gene target) Enhance regulation of 1 target Exogenous system doesn t compete with endogenous factors like RNAi does Good for genes that are lethal if knocked out. LIMITATIONS Target sequence requires adjacent PAM motif Sequence specificity only 14nt long Can recur ~11x in human genome Endogenous chromatin states may prevent binding of dcas9-sgrna complex Influence of genes close to target gene
14 CRISPR-I APPLICATIONS Screen in cell culture or yeast
15 CRISPR-ACTIVATION CRISPR-Activation (CRISPRa) dcas9 fused to activation domain Regulates at transcription level Via transcriptional activator: VP16 x4 = VP64 SUNTAG enhancement
16 CRISPR-A APPLICATIONS Gilbert et al. develop libraries
17 CRISPR IMAGING dcas9 fused to fluorescent marker Compatible with fluorescence microscopy in living cells Unique way to detect chromatin dynamics in cells
18 CRISPR IMAGING CRISPRainbow Multicolor CRISPR imaging Labels multiple loci in living cells Better study chromatin dynamics Methods Orthogonal Cas9s with various fluorophores grnas fused to orthogonal protein-interacting RNA aptamers These recruit RBPs tagged with different fluorescent proteins b627-89d fc/crispr-visualize-horizontal2.png 600x300_q85_crop_subsampling-2_upscale.png
19 SINGLE BASE EDITING What is it? dcas9 + AID (activation-induced cytidine deaminase) What does it do? Converts C T Potential uses Drug discovery Gene disruption Engineering mouse models for point mutation-caused diseases Current limitations Only C T possible right now Dx1_w_h2rwbiPr81HwAAAA&mediaurl=https%3a%2f%2fwww.sciencenews.org%2fsites%2fdefault%2ffiles%2f2016%2f08%2f090316_crispr2_37 0.png&exph=214&expw=370&q=dead+cas9&simid= &selectedIndex=0&ajaxhist=0
20 TEMPORAL CONTROL OF CRISPR What is it? video abstract: A CRISPR on/off switch Use anti CRISPR proteins to shut CRISPR off after editing Natural anti-crispr protein First ones found in 2013 (Bondy-Denomy, Pawluk, Maxwell, Davidson; Nature) Cas9 anti-crispr in 2016 (Cell) AcrIIA4 fits into DNA binding pocket of Cas9 = OFF Anti-anti-CRISPR proteins = ON switch TBDiscovered Uses Temporal/Spatial control of CRISPR enhance therapeutic uses Prevent effects from residual Cas9 og/viruses-keep-crispr-in-check/
21 MULTIPLEXED EDITING What is it? Editing multiple genomic regions using Cpf1 instead of Cas9 Cpf1 discovered by Zhang Lab at Broad Institute How does it work Cpf1 uses smaller guides (40bp instead of 100bp) & contains its own processing machinery Easier to package into lentiviruses Applications Study complex diseases caused by multiple mutations (like cancer)
22 RNA EDITING What is it? REPAIR: RNA Editing for Programmable A to I Replacement How does it work? ADAR(E488Q) fused to Dead Cas13b Benefits? Changes are not permanent in genome Applications ALS, myotonic dystrophy, FXTAS, etc x262_q85_crop_subsampling-2_upscale.png
23 CRISPR EPIGENETIC MODIFICATIONS Fuse to p300, Tet1, DNMT3a
24 ENCHIP WITH CRISPR enchip engineered DNAbinding molecule-mediated chromatin immunoprecipitation Use CRISPR system to isolate specific genomic regions Fuse tag to dcas9
25 GAME CHANGER OR PANDORA S BOX How would you use it? Pick one of the CRISPR applications and apply it to your work. Either undergrad, rotation project, or proposal topic Answer the following questions 1. What application of CRISPR are you using? 2. What question are you using it to answer? 3. Describe your methods in as much detail as possible Controls, test condition, cell or organism, etc. 4. What are caveats to your approach? GNXClJ1mLiGUykX2XwcWk3akrNPoBQm7ih3jMoQw9MG5htA
26 DESIGNING A CRISPR/CAS EXPERIMENT 1. Define your question 2. Choose your Cas 3. Choose your model system 4. Design your guide RNAs (or order libraries from addgene) Order vectors from addgene 5. Pick your delivery system For Cas9 & grna 6. Validate your edit
27 GENETIC MANIPULATION Genetic Manipulation Application Cas9 grna Additional Considerations Knockout Permanently disrupt gene function in a particular cell type or organism without a specific preferred mutation Cas9 (or Cas9 nickase) Single (or dual) grna targeting 5 exon or essential protein domains High-fidelity Cas enzymes increase specificity. Dual-nickase approach increases specificity but is less efficient. Each putative knockout allele must be experimentally verified. Edit Generate a specific user-defined sequence change in a particular gene, Cas9 (or Cas9 such as generating a point mutation nickase); Base editor or inserting a tag Single (or dual) grna targeting the region where the edit should be made HDR requires a repair template and displays reduced efficiency compared to NHEJ knockout. Base editors can make a limited set of mutations. Repress or Interfere (CRISPRi) Reduce expression of a particular gene(s) without permanently modifying the genome dcas9-repressor (such as dcas9- KRAB) or dcas9 grna(s) targeting promoter elements of target gene dcas9-krab is more effective than dcas9 alone for mammalian cell lines. Activate (CRISPRa) Increase expression of an endogenous gene(s) without permanently modifying the genome dcas9-activator (such as dcas9- VP64) grna(s) targeting promoter elements of target gene Many different activators exist, including the multi-plasmid SAM system.
28 DESIGNING GUIDE RNAS Many web platforms Depend on your use CRISPRko CRISPRi/a Etc. crispr.mit.edu Zhang lab at MIT
29 enome_editing/crispr_cas9/resources /Online_tools_for_guide_RNA_design DESIGNING GUIDE RNAS Noteworthy Features of Web-Based sgrna Design Tools Name Graphical user Available species interface Input Output Ranked list CRISPR Design ToolCitation > Yes 15 DNA sequence Candidate guide sequences and off-target loci Yes CHOPCHOPCitation Yes 23 DNA sequence, gene name, Candidate guide sequences and No > genomic location off-target loci CasFinderCitation > No User input DNA sequence Candidate guide sequences and Yes (Perl script) off-target loci Cas- Yes 11 Guide sequence Off-target loci for guide sequences No OFFFinderCitation > FlyCRISPRCitation > Yes 18 DNA sequence Candidate guide sequences and No off-target loci E-CRISPCitation > Yes 31 DNA sequence or gene Candidate guide sequences and Yes name off-target loci Guide RNA Sequence Yes 10 DNA sequence Candidate guide sequences and No Design PlatformCitation > off-target loci CasOTCitation > No (Perl script) User input Guide sequence Off-target loci and additional guide sequences No CRISPR-ERACitation Yes 9 DNA sequence, gene name, Candidate guide sequences and Yes > or TSS location Benchling Yes 5 DNA sequence or gene name distances to TSS Candidate guide sequences and off-target loci YesReferenc es
30 EXPRESSION SYSTEM Expression System Components of System Application Mammalian expression vector Cas enzyme promoter can be constitutive (CMV, EF1alpha, CBh) or inducible (Tet-ON); U6 promoter is typically used for grna May contain reporter gene (e.g. GFP) to identify and enrich positive cells, or selection marker to generate stable cell lines Transient or stable expression of Cas9 and/or grna in a mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and grna can be present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify and enrich positive cells Packaging and envelope plasmids provide the necessary components to make lentiviral particles Stable, tunable expression of Cas9 and/or grna in a wide variety of mammalian cell lines Useful for difficult to transfect cell types. Can also be used in vivo A common choice for conducting genome-wide screens using CRISPR AAV transduction RNA delivery of Cas9 and grna Cas9-gRNA ribonucleoprotein (RNP) complexes 4.5 kb packaging limit (only compatible with smaller Cas enzymes) CRISPR elements are inserted into an AAV transfer vector and used to generate AAV particles In vitro transcription reactions generate mature Cas9 mrna and grna, which are then delivered to target cells through microinjection or electroporation. Purified Cas9 protein and in vitro transcribed grna are combined to form a Cas9-gRNA complex, which is delivered to cells using cationic lipids Transient or stable expression of SaCas9 and/or grna Infects dividing and non-dividing cells AAV is the least toxic method for in vivo viral delivery Transient expression of CRISPR components Expression decreases as RNA is degraded within the cell Short window of CRISPR component expression may decrease offtarget effects Can be used to generate transgenic embryos Transient expression of CRISPR components Short window of CRISPR activity may decrease off-target effects
31 DESIGNING A CRISPR/CAS EXPERIMENT 1. Define your question 2. Choose your Cas 3. Choose your model system 4. Design your guide RNAs (or order libraries from addgene) Order vectors from addgene 5. Pick your delivery system For Cas9 & grna 6. Validate your edit
32 CRISPR HW Look for this next week! Skeath HW due next Thursday
33 RESOURCES Naturally occurring off-switches for CRISPR-Cas9 (Pawluk et al. 2016, Cell)
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