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Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation By Luke A. Gilbert et. al. Presented by: Sabrina Will and Ashley Boydd Background -At the time of this paper, permanently modifying or deleting DNA could be done with customized zinc finger proteins, TALE (transcription activator-like effector) endonucleases, and CRISPR/Cas9 -However, zinc finger and TALE require unique fusion proteins in order to modulate transcription rather than simply knock out a gene, which would be rediculous to try and use for genome-wide screens -Programmed RNAi molecules could be used to knockdown mRNAs, but they had too many off-target effects What is CRISPR? • First Discovered in archaea and later in bacteria; serves as a primitive immune system against viruses • “spacer” sequences are remnants of genetic code from invaders • If spacers are complimentary to a sequence, degradation of the nucleic acid occurs • There are a few types of CRISPR systems but type II (CRISPR-Cas9) is most commonly used Important Components of CRISPR Two main components: • gRNA (guide RNA): contains sequence to be targeted and necessary for cas9 binding • Cas9: acts as an endonuclease Limitations of CRISPR-CAS9 -Causes irreversible frameshift disruptions, meant for knocking out genes, which makes it difficult to study essential genes and lnRNAs - Double stranded breaks can be cytotoxic -Cells can be good at accidentally fixing the damage due to errorprone DNA repair, limiting the ability to knock out all alleles Gilbert’s Previous Study -The year before this paper was published, Gilbert and his team developed a modified CRISPR/Cas9 technology called CRISPRi, which was able to repress genes at the transcriptional level -Worked one of two ways: -1. Directly blocking RNA polymerase activity (dCAS9) -2. Through effector domain-mediated transcriptional silencing (dCAS9KRAB) - They next wanted to better understand and optimize this new tool they had developed Primary Aims • Develop and test a method for high-specificity, genomescale modulation of transcription of endogenous genes in human cells using CRISPRi/a • Aimed to accomplish this in three steps: 1. Perform a saturating screen to test the activity of every unique sgRNA broadly tiling around transcription start sites of 49 genes known to modulate cellular susceptibility to ricin 2. From the screen, extract distinct rules for regions where CRISPRi/a maximally changes the expression of endogenous genes and also for predicting off-target effects 3. Validate these libraries by screening for genes that control cell growth and response to a chimeric cholera/diphtheria toxin Step 1: Saturating screen • Used massively parallel oligonucleotide synthesis to generate a library of sgRNAs that tile a 10kbp window around the TSS of 49 genes known to contribute to ricin resistance • 54,810 total sgRNAS • The entire library was transduced into K562 human myeloid leukemia cells stably expressing dCas9 or dCas9-KRAB using lentiviral particles • Then used deep sequencing using the sgRNAs as barcodes to count the frequency of each sgRNA after growing cells in either standard conditions or with exposure to ricin CRISPRi Tiling Screen • Define a set of rules for construction of a genome scale CRISPRi library • Use ricin resistance phenotype to measure transcriptional repression CRISPRi Tiling Screen (cont.) • Compare the phenotype strength of CRISPRi with previously published shRNA data • CRISPRi shows much stronger phenotype in most cases than shRNA CRISPRi Tiling Screen (cont.) • Took 30 sgRNAs and created mismatch base pairing • CRISPRi activity dramatically decreased with just a single mismatch • Shows CRISPRi results in high specificity with very little off target repression activity CRISPRa Tiling Screen • Developed new CRISPRa method termed sunCas9 • One sgRNA with one binding site is sufficient to activate transcription • dCas9 fusion protein bound to DNA recruit multiple copy of activating effector domain • What are the optimal conditions? CRISPRa Tiling Screen (cont.) • Ricin resistance phenotype peaks downward closest to the TSS, demonstrate CRISPRa actually works! • Implies similar to CRISPRi, CRISPRa works best near the start of TSS Step 2: Extracting Rules for optimal use of CRISPRi/a for genome-wide use From CRISPRi/a tiling screens, were able to extract rules for optimal use of both systems: • CRISPRa works best −400 to −50 bp upstream from the TSS while CRISPRi works best −50 to +300 bp relative to the TSS of a gene • CRISPRi/a are both extremely specific systems that result in very little off targeting effects Step 3: Validating Libraries by Screening • Gilbert and his team demonstrated the application of their CRISPRi/a technology in two ways: 1. Screening for genes that control cell growth 2. Screening for genes that govern a response to a CholeraDiphtheria fusion toxin (CTx-DTA) Screening for genes that control cell growth • First screened for genes essential in cell growth of K562 cells • Showed that no off target effects were happening in K562 cells; Y chromosome and Olfactory genes unaffected • Demonstrated no toxicity occurring under these conditions Screening for genes that control cell growth (cont.) • Synthesized and Cloned genome-scale CRISPRi sgRNA library targeting 15,977 human protein coding genes • Used metric of average growth phenotype to identify hit genes • Validating Gilbert’s approach as a screening platform. Revealing Response Pathways for a Cholera-Diphtheria Fusion Toxin from a CRISPRi/a Screen A. Model for CTx-DTA binding, retrograde trafficking, retrotranslocation, and cellular toxicity B. Overview of top hit genes detected by CTx-DTA screen. Dark red/blue circles represent top 50 sensitizing and protective hits, light red/blue circles represent other proteins that fall into the complexes 1. Red= causes sensitivity to toxin, Blue= reduces sensitivity to toxin, Stars= previously identified by haploid mutagenesis screen 2. Circle area is proportional to phenotype strength C. CRISPRi/a hits in sphingolipid metabolism, which is responsible for toxin uptake Overall Conclusions • CRISPRi/a results are highly reproducible • Any intrinsic toxicity is undetectable • Transcriptional repression using CRISPRi is inducible, reversible, and can target essential genes • CRISPRi/a can be used to control transcript levels for endogenous genes across a broad range • Properly designed CRISPRi reagents are highly specific Criticism • Collections of figures were disjointed and out of order; should have dispersed figures throughout text as opposed to lumping them all into a page • Lots of information; felt that some experiments should have had their own papers Future Readings • CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology by New England BioLabs • CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154:442–451 by Gilbert et al. References • Gilbert et al. 2014. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell; 159:647-661. • Gilbert et al. 2013. CRISPR-Mediated modular RNA-guided regulation of transcription in eukaryotes. Cell; 154, 442-451.