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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
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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
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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 CRISPRs, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, were first discovered in archaea (and later in bacteria). CRISPRs serve as part of the bacterial immune system, defending against invading viruses. They consist of repeating sequences of genetic code, interrupted by “spacer” sequences – remnants of genetic code from past invaders. Later, if the cell encounters foreign DNA, it will compare it to the short stored sequences of the spacers. If the sequences of the stored and foreign DNA match, the foreign DNA will be destroyed by an enzymes, one of which is known as cas9.
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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 CRSIPR requires two main components in order to function properly; a guide RNA and the cas9 enzyme. The gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified. What PAM sequence is (if asked): The PAM sequence is absolutely necessary for target binding and the exact sequence is dependent upon the species of Cas9 (5′ NGG 3′ for Streptococcus pyogenes Cas9).
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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 error-prone DNA repair, limiting the ability to knock out all alleles
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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 (dCAS9-KRAB) - They next wanted to better understand and optimize this new tool they had developed
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Primary Aims Develop and test a method for high-specificity, genome-scale modulation of transcription of endogenous genes in human cells using CRISPRi/a Aimed to accomplish this in three steps: 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 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 Validate these libraries by screening for genes that control cell growth and response to a chimeric cholera/diphtheria toxin Currently, the dominant tool for programmed knockdown of mRNAs is through a method known as RNA interference (RNAi). However, RNAi has pervasive problems with off-target effects, which can be detrimental in the context of large-scale screens. Other gene editing techniques utilizing the zinc finger and TALE proteins has also been able to successfully silence/repress genes; however such methods require that each transcript target needs a unique fusion protein, which makes utilizing these methods to genome-scale is arduous. As such, Gilbert wanted to identify the advantages of using CRISPR as opposed to RNAi and zinc fingering.
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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
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CRISPRi Tiling Screen To explore the limits of CRISPRi’s repression of endogenous genes 49 genes that were previously shown to modulate cellular susceptibility to the AB toxin ricin were targeted. The extent of gene repression for these genes typically has a monotonic relationship with the ricin-resistance phenotype, allowing us to use a ricin-resistance score calculated by monitoring sgRNA frequencies in a pooled screen to indirectly measure transcriptional repression. CRISPRi can repress transcription by directly blocking RNA polymerase activity (dCas9) or through effector domain-mediated transcriptional silencing (dCas9-KRAB) Plotting this data for all 49 genes showed that active sgRNAs cluster around or just downstream from the TSS (transcription start sites) of each gene for dCas9-KRAB and dCas9, respectively. Strong CRISPRi activity is obtained by targeting dCas9-KRAB to a window of DNA from −50 to +300 bp relative to the TSS of a gene, with a maximum in the ~50–100 bp region just downstream of the TSS. This suggested that optimal activity leverages the combined activity of dCas9 interference along with repression from the KRAB domain. We saw that strong CRISPRi activity is obtained by targeting dCas9-KRAB to a window of DNA from −50 to +300 bp relative to the TSS of a gene, with a maximum in the ~50–100 bp region just downstream of the TSS Define a set of rules for construction of a genome scale CRISPRi library Use ricin resistance phenotype to measure transcriptional repression
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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 In this screen, Gilbert and his team wanted to compare the CRISPRi technology with that of shRNA. The figure displays the 49 genes utilized and the corresponsing measurements of their phenotype. In virtually every case the normalized ricin phenotype z-score or p-value is stronger (in many cases far stronger) than seen with a comparably-sized shRNA library (generated by sub-sampling our published data)
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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 To assess CRISPRi off-target activity at endogenous genes, we selected a set of 30 sgRNAs from our tiling library (6 sgRNAs/gene targeting 5 genes). For each of these sgRNAs, we tested the activity of a series of derivative sgRNAs with a variable number and position of mismatches. Gilbert was able to measure the relative amount of gene repression for sgRNAs with or without mismatch base pairing targeting the same DNA locus. We found that even a single mismatch at the 3′ end of the protospacer decreased CRISPRi activity on average, while combinations of mismatches that pass our off-target filter abolished activity
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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? We recently developed an improved CRISPRa method, termed sunCas9, in which expression of a single sgRNA with one binding site is sufficient to robustly activate transcription (Tanenbaum et al.) In the sunCas9 system, a single dCas9 fusion protein bound to DNA recruits multiple copies of the activating effector domain, thus amplifying our ability to induce transcription
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CRISPRa Tiling Screen (cont.)
Similar to the CRISPRi tiling screen. Gilbert targeted genes capable of modulating cellular sensitivity to ricin to learn more about his new CRISPRa system. They first transduced K562 cells stably expressing the sunCas9 system with the sgRNA tiling library and screened for ricin phenotypes using the same methodology for CRISPRi. Analysis of data for individual genes or averaged data for all 49 genes demonstrated that many sgRNAs for each gene affected ricin resistance. Our negative control sgRNAs showed very little activity and were not correlated between biological replicate screens, suggesting that CRISPRa activity is specific. We observed a peak of active sgRNAs for CRISPRa at −400 to −50 bp upstream from the TSS. 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
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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
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Step 3: Validating Libraries by Screening
Gilbert and his team demonstrated the application of their CRISPRi/a technology in two ways: Screening for genes that control cell growth Screening for genes that govern a response to a Cholera- Diphtheria fusion toxin (CTx-DTA)
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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 To conduct this study, they followed the rules dictated by their previous CRISPRi tiling screen and chose a library size of 10 sgRNAs/gene. Over half of the sgRNAs conforming to these rules gave clear ricin phenotypes. For a library with 10 sgRNAs/gene, 94% of the genes would thus have 2 or more highly active sgRNAs. To explore the prevalence of off-target effects, we examined two classes of genes not expected to show any on-target activity in our screen: olfactory receptors and genes on the Y chromosome. The sgRNAs that target these genes were designed and picked in the same manner as the rest of library; however, olfactory receptors should not be expressed in this cell type and, as K562 cells are derived from a female donor, sgRNAs that target genes on the Y chromosome lack a DNA target. As with the negative controls, these genes show no phenotype on average and exhibit very little correlation between replicates (Spearman R = for olfactory genes and −0.052 for Y-targeting). Also observed no evidence of non-specific toxicity due to expression of dCas9-KRAB and our sgRNA library in K562 cells suggesting that dCas9 bound to the genome is not toxic under these conditions
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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. Synthesized and cloned a genome-scale CRISPRi sgRNA library targeting 15,977, human protein-coding genes. To identify hit genes in this screen, we used a metric of average growth phenotype (γ) for the top three sgRNAs for each gene. Among the top hits were genes involved in essential cellular functions, including translation, transcription and DNA replication
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Revealing Response Pathways for a Cholera-Diphtheria Fusion Toxin from a CRISPRi/a Screen
Model for CTx-DTA binding, retrograde trafficking, retrotranslocation, and cellular toxicity 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 Red= causes sensitivity to toxin, Blue= reduces sensitivity to toxin, Stars= previously identified by haploid mutagenesis screen Circle area is proportional to phenotype strength CRISPRi/a hits in sphingolipid metabolism, which is responsible for toxin uptake This is what was previously known about CTx-DTA uptake, which was very little. A screen similar to the one performed for cell growth revealed the pathways and enzymes involved in uptake and response to this toxin. It was revealed that sphingolipid metabolism contributes to the uptake of the toxin. Inhibiting some genes (left half of the circles) and activating some others (right side of the circle) makes a cell more vulnerable to taking in the toxin.
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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
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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
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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.
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References Gilbert et al Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell; 159: Gilbert et al CRISPR-Mediated modular RNA-guided regulation of transcription in eukaryotes. Cell; 154,
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