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Screens check biological functions in systemic ways. Completes a wide scan to see which genes are involved in specific activities instead of specific genes each time by scanning the whole genome. This requires advanced machinery. HTS (High-throughput screens) subject many biological samples to simultaneous testing under given conditions. It used to only happen in yeast, but now mechanisms such as CRISPR make it possible in mammalian cells as well. Often uses microscopes and specific robotics. 

There are two options for HTS screening: genetic screening effects genes (RNAi, CRISPR), and drug screens (drugs, small molecules) that effect the genes. 

Genetic screens Silencing screens include siRNA, shRNA, CRISPR-KO, and CRISPRi. Overexpression genes include cDNAs (add additional gene segment for increased expression) and CRISPRa. 

RNAi is a defense mechanism against viruses found mainly in plants and drosophila, more recently they found it in mammalian cells as well. Our bodies are not used to seeing long dsRNA in the cytoplasm (ssRNA is more common). The presence of dsRNA signifies a pathogen, and so we want to protect ourselves by cutting it into smaller fragments. This cutting is done by the RISC complex. If mRNA fragments are found in the cell that match these fragments, they are silenced or cleaved. We use it artificially to silence cells, using a primer for the gene we want to silence. This was first shown in C. elegans: dsRNA introduced was able to degrade a specific mRNA. This RNAi was systemic, meaning dsRNA introduced in one tissue led to gene silencing in other tissues. dsRNA was inserted by soaking worms in dsRNA solution, injection, feeding, or generating transgenic hairpin-expressing animals. 

Unlike with C. elegans, inducing this in mammalian cells may cause immune reactions. This can be avoided by adding smaller RNA molecules to begin with (23-25 bp siRNA) that don’t alert the immune system, with many modifications that prevent reactions. It is as though these smaller fragments have already been processed by the cell and  silenced by the immune system. Alternatively, adding dsRNA is a possibility, as immune reactions do not always occur. Lastly, shRNA (DNA plasmid) can be entered and transcribed to RNA, allowing RNAi activity. Generally, RNA enter better into cells, creates more copies, and silences better. However, they are not transcribed during cell division and so effect will deplete over time. shRNA can be engineered to enter the genome and stay forever as DNA molecules, so that the gene will always be silenced. siRNA is RNA, shRNA is a plasmid (DNA) that is reverse transcribed to get RNA. 

siRNA is transfected into cells using a lipid base that wraps around the RNA, protecting it, while also permeating the membrane. shRNA can also be transfected (won’t enter genome), or use viruses to insert into the genome to procure constitutive expression. 

Review of CRISPR. CRISPR sends Cas9 to cut specific points in the genome, led by gsDNA. A complex of gsDNA:Cas9 reaches the genome, and then Cas9 has helicase activity to cut both strands of DNA (DSB), and then the cell uses its natural mechanisms to fix the break. If the break isn’t properly corrected there can be insertions, deletions, inversions, etc. If the reading frame moves, a proper protein will not be produced. This allows us to entirely silence genes (in contrast with RNAi, which works on the RNA level and cannot entirely silence proteins). CRISPR can silence a much higher percentage of genes since it is at the DNA level. CRISPR can also be used to silence different genes to check what effect this will have on an organism. 

CRISPRi allows us to check the function of important genes that we cannot completely knock out. It is more specific than RNAi and uses CRISPR technology so is better. The CRISPRi system is directed to the promoter. Cas9 is a huge protein that will bind strongly to the promoter and block transcription machinery (RNApol) from transcribing the gene. Additionally, Cas9 is suited with transcriptional repressors, which prevent initiation factors for transcription from starting transcription. The Cas9 used here does not cut; we used dCas9 which is inactive. 

CRISPRa works by adding transcription factors to Cas9, which enhance transcription. In this case, Cas9 is located so that it can recruit transcription factors without blocking the promoter. It sits a bit further away from the promoter than in CRISPRi. This is endogenous, so is limited to the existing promoter. Adding cDNA can increase expression of a specific gene. It is entered via transfection of a plasmid or with viruses. It is usually a strong promoter that increases enhanced gene activity. Here, the promoter itself can be changed to be made stronger. 

cDNA libraries are very expensive. They have a ton of genes and have to include genes from a lot of different sources to make sure they are all present. Some labs worked together to make joint libraries and save themselves money. 

Regular cells show specific expressions when both genes are functional. For CRISPR-KO, +/+ has nothing changed and so stays the same as regular cells. If one is knocked out, +/-, half of the product is received. If it is on both cells, -/-, we won’t get any expression. 

RNAi is somewhere in the middle. cDNA/CRISPRa will overexpress genes. CRISPRa is more specific than cDNA. 

Screens can be arrayed, where the contents of every plate is known (cDNA/CRISPR libraries). This method is more expensive, work is harder and requires a reader using a microscope or measurement of how many cells are divided or other phenotype. The advantage is that the answer is immediate; the identity of the gene is known right away. Pooled screens take a group of cells in a mixture and infect them with viruses to give a single genetic manipulation. The cells are examined together, with each cell presenting a different silencing/over expression. Then, examine חיות or add a reporter gene (ex: DNA damage will be green) and see which genes affect DNA damage. This is much simpler, cheaper, but is limited in terms of analytical options available. Pooled for constitutive silencing can be induced with viruses (gRNA or shRNA - not RNAi). RNAi can only be done in a pooled array. shRNA and CRISPR can be done in a pulled array. 

To plan a screen, first identify a gene (can also be genome-wide). Create a library with gRNA against these genes (smaller is better). Establish what biological activity you want to check (damage to DNA, protein, etc) and decide on a pulled/arrayed screen. For example, take a reporter gene that lights up (luciferase) when a gene is expressed. If it is put with stress proteins, you will see which genes have more stress. Can see location in cells, expression, etc and how this impacts cell phenotype. This is mainly for arrayed screens. Pulled is only for cell growth or with a reporter gene. 

An example of a screen is for DAP (death associated proteins), which have programmed cell death in response to IFN-𝞬. Screened RNAi and silenced all genes to see which genes could be silenced to prevent the cells from dying in response to IFN-𝞬 (pro-apoptotic genes that were inhibited no longer led to apoptosis). Very few cells with KO survived, these cells lacked DAPs. 

Another example is with corona, a pulled screen that checks which genes help the virus to proliferate. Insert library into cells and then infect with the virus. See how much gRNA was in the cell before and after and sequence (next generation sequencing). If a gene stops the virus, it will be found in surviving cells at high concentration. The largest target was the receptor for the virus (but this cannot be silenced since it is needed by the cell for other purposes). 

If we want to do a screen but don’t have a library and don’t want to screen the whole genome, we can send a lab a list of the specific genes we want to synthesize (gRNA or cDNA). Pulled screens will always be done with viruses to make sure cells each got one modification. Observe cells over time and examine the number of gRNA at start and after to see how the gene silencing impacted the cell. Use NGS to sequence and determine what modification was made in the cells. CRISPRa is advantageous than CRISPR-KO for essential genes, and that CRISPR-KO is mainly for protein-coding genes and CRISPRa can work with noncoding genes (majority of genome). 

Drug screens are done in arrayed screens. Unlike genetic screens, we don’t know what the target is, only that a specific drug has an intended effect without harming cells. For example, copaxone (treated MS) created mouse models without knowing exact molecular models with which the drug acted. This is different from gene targets, for which we need to identify the exact mechanism. Usually do a screen of very high concentrations of many drugs, and then select effective drugs and do alterations of them. Leishmania doesn’t have a good cure; researchers tried to create a cream to fix it. They screened a lot of drugs (3 millio compounds)  for infected mice, and from there identified the molecular pathway.