Perla and Q will be presenting next week the following papers and topic on using CRISPR/Cas9 screens:
Daniloski Z, Jordan TX, Wessels HH, Hoagland DA, Kasela S, Legut M, Maniatis S, Mimitou EP, Lu L, Geller E, Danziger O, Rosenberg BR, Phatnani H, Smibert P, Lappalainen T, tenOever BR, Sanjana NE. Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells. Cell. 2021 Jan 7;184(1):92-105.e16. doi: 10.1016/j.cell.2020.10.030. Epub 2020 Oct 24. Xiong, X, et al., CRISPR/Cas9 for Human Genome Engineering and Disease Research. Annu Rev Genomics Hum Genet. 2016 Aug 31;17:131-54. doi: 10.1146/annurev-genom-083115-022258. Epub 2016 May 23.
16 Comments
Dianna Xie
2/17/2024 02:18:08 pm
In the discussion session of “Identification of Required Host Factors for SARS- CoV-2 Infection in Human Cells”, is it referring that drugs can help block the entry of the virus to the host by altering the receptors?
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Rohan Babaria
2/20/2024 06:57:25 pm
In the CRISPR/Cas9 for Human Genome Engineering and Disease Research article they mention that even when targeting for HDR pathways it still only happens around 33% of the time. I wanted to know what may be some of the side effects associated with those errors as NHEJ can introduce/delete key parts of the sequence.
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Rebecca Wright
2/20/2024 11:22:32 pm
In the "Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells" paper, why did the authors choose a lung cancer cell line as a model? Wouldn't it have been better to use a non-cancer cell line due to the various mutations and epigenetic modifications that cancer cells have?
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Julia Carey
2/21/2024 04:58:47 pm
For the Western Blots the authors performed in Figure S3A, does the band need to be completely absent to signify Cas9 mediated loss-of-function? Or is a light band still present acceptable validation?
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Sara Fritz
2/21/2024 09:01:35 pm
I understand that the use of lung cells in the CRISPR screen was probably due to the virus being respiratory and that lung tissue would have a strong connection with SARS-CoV-2 infection. I don’t understand how they said that the genes implicated in their findings are broadly expressed across many tissues and that this could mean that the mechanisms are not tissue-specific. Why do the genes they found tend to be broadly expressed and what does this mean for the research? If a different tissue was used would we find tissue specific genes implicated?
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Joely Swanson
2/21/2024 09:12:50 pm
Why did they use cancer cells for this study? Do these cells accurately reflect how Covid acts in noncancerous cells?
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Samuel Kivi
2/22/2024 12:02:37 am
The Results section of "Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells" mentions that the genes associated with regulation of LDL cholesterol (sometimes referred to as the "bad" type of cholesterol) are involved in SARS-CoV-2 infection. Could these results suggest that lowered cholesterol levels help reduce infection rates of SARS-CoV-2?
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Zoe Zwick
2/22/2024 12:09:32 am
In the paper, "Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells", they mention that there may be cell-type specific responses. What cell types do you think would most likely have differential genetic circuits; which cells do you think would be the most interesting to look at in future studies?
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Brooke Fuerstenau
2/22/2024 12:22:34 am
In the "CRISPR/Cas9 for "Human Genome Engineering and Disease Research" paper they talk about RNAi and how it is used less due to off-target effects, how is CRISPRi designed so that it doesn't have these same affects?
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Michelle Conte
2/22/2024 12:42:28 am
When the paper discusses cross referencing the genes of interest from the original genome loss of function CRISPR experiment, they talk about testing inhibitors on a selection of druggable genes that have known and tested inhibitors. Are all of these inhibitors to gene expression, protein formation, or do they result in some sort of degradation of a gene's protein?
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Miles Giehtbrock
2/22/2024 01:00:48 am
Why is Cas9 the "gold standard" enzyme used if different cas type proteins are found all over archaea and procaryote genomes? Is it just because it was the first to be discovered? I know cas12 also exists. Again why do we use cas9 for nearly everything and not other orthologous proteins that may be better suited for genome engineering?
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2/22/2024 07:12:01 am
In “Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells,” what does it mean for a virus to be a positive-sense RNA virus?
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2/22/2024 09:47:05 am
For "Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.", how do the identified host factors for SARS-CoV-2 infection compare to those required for other coronaviruses, like SARS-CoV and MERS-CoV
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Madeline Blum
2/22/2024 11:46:26 am
How did they determine which MOI values reflected this SARS-Cov-2 strain's infection rates (0.01 for low, 0.3 for high)? They did not complete an additional assay, and no background information was cited that 0.3 is a high infection level.
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Kate Stack
2/22/2024 11:49:37 am
The primary paper discusses tamoxifen as an inhibitor to ultimately reduce viral load. In my research lab we sometimes will use tamoxifen to alter the gut microbiome composition. There are a lot on interactions between the microbiome and the immune system resulting in part from metabolites. I wonder if by introducing tamoxifen as a treatment would have detrimental side effects to the immune system functioning? Even if the COVID-19 viral load is reduced, the loss of potentially useful bacteria could impair an effective immune response.
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Praneeth Venigalla
2/22/2024 01:23:23 pm
How do the specific host factors for SARS-CoV-2 infection identified by Daniloski et al. (2021) contribute to our understanding of virus-cell interactions, and how might CRISPR/Cas9 technology, as discussed by Xiong et al. (2016), be applied to manipulate these interactions for therapeutic purposes?
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