Genome-Scale CRISPR-Mediated Control of the Gene Repression and Activation Luke A. Gilbert, Max A. Horlbeck, Britt Adamson, Jacqueline E. Villalta, Yuwen Chen, Evan H. Whitehead, Carla Guimaraes, Barbara Panning, Hidde L. Ploegh, Michael C. Bassik, Lei S. Qi, Martin Kampmann, Jonathan S. Weissman By: Navjot Naur & Yazmin Rodriguez

Alter transcription of endogenous genes using CRISPRi/a Overview Alter transcription of endogenous genes using CRISPRi/a tested activity of sgRNA around transcription start site of genes known to initiate cellular response to ricin (toxic protein) extracted regions where CRISPRi or CRISPRa changed the expression of genes algorithm to design two genomic libraries testing genes with sgRNA

Background CRISPR Cas9 Defense mechanism used in bacteria Has two components: guide RNA and Cas9 endonuclease Guide RNA consists of CRISPR RNA and tracr RNA can be used to cut any DNA sequence at a precise location pnabio.com CRISPER Cas9 is a naturally occurring defense mechanism used in bacterium to protect themselves from infection caused by viruses. When the bacterium detects the viral DNA, it produces two types of short RNA. These two RNAs from a complex with a protein called Cas9 Cas9 is a nuclease that can cut dna One RNA contains a sequence that matches that of the invading virus. This is the guide RNA. when the matching sequence finds its target on the viral gemone, the cas9 cuts the dna short guide RNA consists of CRISPER RNA and tracr RNA: a duplex which is used to target the cas9 nuclease can be used to cut any DNA sequence at a precise location not just in a test tube but living orgainsms

CRISPR Cas9 Introduction naturally occurring process used bacteria engineered mechanism: researchers have fused the two RNA’s together to form a single guide RNA that serves the same function by adding a hairpin loop to the duplexing site. this is the mechanism used in the paper. the cleavage induces a mutation which causes malfunction in the expression of the gene. If the mutation occurs, this will no longer be recognized by the complex, however if the mutation does not occur the complex will return until the mutation occurs. In addition the CRISPR cas9 can be encoded by transfecting it into cell lines or carried in a viral vector. the most common viral vector used is the lentivirus (subset of retroviruses)

Experiment inject short guide RNA into lentivirus which infect K562 cells that already express cas9. T he sgRNA is designed for the 49 genes that are susepitible to ricin. There are 1000 short guided RNA per 10kb area near the transcription strat site of each gene. After the infection they treat the cell with ricin, and the untreated are the control. The 49 genes are known to repress ricin (toxin).

CRISPRi / CRISPRa dcas9: catalytically dead version of Cas9 dCas9-KRAB: catalytically dead version of Cas9 fused to a transcriptional silencer (KRAB domain) dCas9-SunTag: recruit transcriptional activators co-developed a screening platform using a catalytically dead version of Cas9 (dCas9) to recruit either a transcriptional repressor domain (CRISPRi) or transcriptional activator domains (CRISPRa) to specific DNA loci close to the transcription start site (TSS) of mammalian genes. They determined rules for sgRNA design and targeting that enable reversible repression or activation of gene transcription. Based on these rules, they constructed genome-wide sgRNA libraries optimized for either CRISPRi or CRISPRa pooled genetic screens. CRISPRi-based loss-of-function screens and CRISPRa-based gain-of-function screens yield rich, complementary insights into cellular pathways. Dcas9: and sgrna and makes its way to the gene but Dcas9 krab: sites at the transcription site and inhibits the polymerase from transcribing the gene kampmannlab.ucsf.edu

when knocked down by RNAi. Ricin-resistance phenotypes, comparing CRISPRi and sgRNAs for genes previously established to cause ricin-resistance phenotypes when knocked down by RNAi. They then synthesized a series of small guide RNAs (sgRNAs) to target dCas9-KRAB to within a 10 kilobase region for each of 49 genes that modulate cellular sensitivity to ricin

median sgRNA activity in a defined window for all genes. Green line: median sgRNA activity in a defined window for all genes. Orange region: observed average window of maximum CRISPRi activity A pooled screen followed by deep sequencing identified the sgRNAs that reproduced the phenotypes previously observed by RNAi, allowing the group to define rules for sgRNA activity and specificity. Analysis of all 49 genes targeted in a tiling sgRNA library. Maximum repression occurred 50–100 nucleotides downstream of the transcription start site (TSS). The group demonstrated how sensitive CRISPRi activity is to mismatches, providing the basis for designing a highly specific genome-wide CRISPRi library. RNA polymerase doesn’t bind at the start site, it binds at the promoter (depending on the gene) Comparing the orange window of the dcas9 krab has a larger widow than dcas9. krab works better. The guide RNA should be designed downstream to the start site because they cause the maximum repression. So after this mutation at the start site Most ricin phenotype was observed then they were bound downstream to the start site because that’s where gene repression occurred. They were able to quantify the abundance of sgrna based on its resistance to ricin compared to contro that received no toxin. These genes are sensitive to ricin thus the inhibition will reade to resistnace thus more aboundace of sgrna because they grow.

dCas9-SunTag sgRNA system for CRISPRa As with the CRISPRi tiling screens, set of rules that allowed construction of a genome-scale CRISPRa library. Many of these rules overlapped with those of CRISPRi (e.g.,sgRNA length and sequence preferences). They were able to monitor the overexpression of the through the single chain antibodies.

median sgRNA activity in a defined window for all genes. Top: sgRNAs targeting VPS54 Green line: median sgRNA activity in a defined window for all genes. Orange region: observed average window of maximum CRISPRa activity negative values represent opposite results than from knockdown observed a peak of active sgRNAs for CRISPRa at _400 to _50 bp upstream from the TSS. CRISPRa, in which an sgRNA directs dCas9-SunTag to recruit an array of transcriptional activators to drive overexpression of a specific locus. Activator works best upstream to start site, most amount of phenotype changes were recodered when sgrna was bound to this site. What you see at the top they did this for every single gene, so this is showing the combined data average. Overexpression of this gene will make you less resistance to ricin thus is it’s negative

CRISPRi knockdown and CRISPRa activation of the same gene can have opposing effects on ricin resistance The same gene is repressed and overexpressed, they have opposite effects on ricin resistnace. Inhibiting leads through CRISPRi leads to expression increase in resistance and overexpression leads to degrese in resistnace thus the sgrna is the negative.

Coexpression of sgRNAs and dCas9-KRAB or dCas9-SunTag is not toxic in K562 cell lines over 16 days fraction of cells expressing sgRNAs and the sunCas9 system was stable over the course of the experiment, indicating that here was no general toxicity associated with the CRISPRa platform (Figure 4B). These data suggest that, like CRISPRi, CRISPRa is specific and nontoxic. Same for dCas9- KRAB . Thus, CRISPRi is highly specific and nontoxic. They used puromyosin to select for cells that have both sgRNA and CRISPR

What they found Control of transcript levels for endogenous genes across a high dynamic range (up to ~1000-fold) reveals how gene dose controls function Mapping of complex pathways through complementary information provided by CRISPRi and CRISPRa CRISPRi provides strong (typically 90%–99%) knockdown of both protein coding and non-protein coding transcripts with minimal off- target activity CRISPRi is inducible and reversible, and can allow for the study of essential gene functions

Application probe the biological roles of all genes within the genome in a single experiment Reveal mechanisms by which cancer cells develop resistance to anti-cancer drugs Identify cellular targets of new drugs Identify tumor suppressor genes that inhibit the growth of cancer cells Identify genes that regulate tissue development