Questions tagged [crispr]
Cluster Regularly Interspaced Short Palindromic Repeats; Naturally a prokaryotic adaptive immune system, elements of CRISPR are increasingly being used for sequence specific DNA targeting in biotechnological applications, especially genome editing. This tag can be used for questions about its natural function or biotechnological applications, including elements of the CRISPR system such as Cas9 and Cpf1 nucleases.
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Can several genes be edited at once and what is the name of that procedure?
In general, if there are multiple genes of interests, or edits at multiple points along a large gene are desired, can several edits done at once (or as many as is possible while minimizing off-target ...
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Are organisms that are genetically modified with a deletion classified as "transgenic"?
Imagine you delete a genomic region in a zygote with a CRISPR plasmid to generate an F1 animal with a genomic deletion.
The animal should be considered transgenic because of the presence of the ...
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Linearising plasmids for CRISPR experiment
I am currently designing a mock CRISPR knock-out experiment, and I’m wanting to insert a plasmid for selection. Using a restriction enzyme at 2750bp for cutting, would the location of the cut site ...
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CRISPR-Cas9 system, DNA repair
As a critical stage in the CRISPR-Cas9 system, two different mechanisms of DNA repair can occur in the target DNA after RNA has been introduced: non-homologous end joining (NHEJ) and homologous ...
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Single-cell subcloning of BV-2 cells?
I am trying to knock-out genes in BV-2 cell line. However, the majority of protocols require cell subcloning and expanding. I tried to subcone these cells and grow clones in DMEM+10% heat-inactivated ...
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How does an engineered supressive Lethal (when present in 2 copies) gene drive spread through a population until causing population extinction?
I understand that at the molecular level a CRISPR mediated gene drive works by copying the altered gene (and the drive containing CAS enzyme, and guide RNA) into the other chromosome containing the ...
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CRISPR/Cas9: How can inserted DNA be used as a donor for the homology-directed repair if Cas9 only creates blunt ends?
The CRISPR/Cas9 method allows new genes to be inserted. After Cas9 cuts the Target-DNA, it can use a homologous piece of DNA as a donor template for homology-directed repair. But HDR only occurs when ...
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Can the genetic sequences for CRISPR components be inserted into the host genome so that the cell perpetually produces CRISPR components?
Theoretically speaking, can you insert the gene sequences for cas9, sgRNA, and promoters into the host genome so that the cell perpetually produces CRISPR components?
In this scenario, I'm guessing ...
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How are spacer sequences created in a prokaryotic genome?
The CRISPR/Cas defense mechanism uses spacer sequences between palindromic repeats to search for the sequence to cut by an endonuclease. But how are these spacers created?
Let's take Bacteriophages, ...
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How many cuts are done during CRISPR-Cas9 in one cell?
In a CRISPR-Cas9 experiment, the protein cuts the site matching the cRNA part of the gRNA. My question is: How many cuts are possible if multiples sites matching the cRNA are found in the cell?
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What are the differences between dual vector CRISPR/Cas9 lentiviral plasmids Lenti‐Cas9‐2A‐Blast and lentiCas9-Blast?
There are multiple widely-used plasmids for using CRISPR/Cas9 with a dual lentiviral vector strategy (Cas9 & sgRNA on different vectors) in mammalian hosts with a Blasticidin selection selection ...
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Is CRISPR mediated RNA editing less specific and less efficient than DNA editing?
According to this diagram, the high efficiency and the high specificity of CRISPR lies in its reversible binding with the target DNA. The Cas protein unzips the target DNA and have the gRNA to base ...
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Would viral diversity result in a change in the effectiveness of CRISPR systems in a population of bacteria, within a closed system?
I have here my hypothesis, does this make scientific sense? Assume this situation is occurring in a closed environment with only bacteria and bacteriophages.
The effectiveness of CRISPR/Cas9, being an ...
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Do bacteria duplicate the copied virus DNA to put into a Cas9 protein when fighting the virus off again?
When bacteria insert a part of invading virus DNA into its own genetic sequence, on the 2nd invasion does it duplicate that copied DNA again from wherever the bacteria placed it and put it into a Cas9 ...
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Can CRISPR knockout of a gene be referred gene inhibition?
Can gene knockout (e.g. by CRISPR) be referred to as gene inhibition? I am trying to use precise words for a manuscript. We first showed siRNA of a gene has effect X. We also found knockout of the ...
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Can we cure cancer with CRISPR dead Cas?
Here's a silly idea I had this morning:
Sequence a bunch of normal patient cells.
Sequence a bunch of tumor cells from a biopsy.
Find a DNA sequence that we're reasonably certain exists in the cancer ...
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Does ribonuclease processing of pre-crRNAs happen co-transcriptionally?
I understand CRISPR-mediated bacterial immunity to occur in the following simplified steps:
A CRISPR array is transcribed from promoters in the leader sequence to yield a precursor CRISPR RNA (pre-...
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Is CRISPR being utilized when scientists use the CRISPR/Cas9 system to edit genes?
"CRISPR" and "Cas9" are different things. When a virus attacks a bacteria, the bacteria stores the viral code of the virus in CRISPR. And when the virus attacks again the Cas9 ...
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How was gene knock out done in pre CRISPR era?
I am trying to understand how CRISPR has made the gene knockout or gene editing process simpler to make transgenic animals. Here is an old (pre CRISPR) flowchart from Manis, 2007 that shows how ...
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What obstacle(s) would be most prevalent in an attempt to use virotherapy to inject Cas into cells to target a cancerous mutation?
Let's say a human cell mutates cancerously, we identify the mutated gene sequence and use CRISPR to mutate the patient's cells to include some variation of the cas genes and spacer sequences matching ...
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When does the Cas9 nuclease stop?
If I understand correctly, the steps of gene editing with CRISPR-Cas9 are roughly as follows
Cas9 nuclease and guide RNA form a complex.
Cas9+guide RNA complex scans genomic DNA and recognizes the ...
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if an SNP were edited using CRISPR What are the chances that, absent artificial selection, wild type alleles would reemerge?
I am researching a fatty acid amide hydrolase (FAAH) SNP RS324420 and FAAH out microdeletion that together lead to reduced pain sensitivity and reduced anxiety (Moreira et al 2008).
The causative ...
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Can we change the Eye/Hair color by knocking out the OCA2, HERC2 and MC1R genes using CRISPR in an adult human?
This paper seems to describe the use of a plasmid delivered by a gene gun to depigment rat skin;
https://www.nature.com/articles/3302264
Published: 27 May 2004
Seeing the gene therapy: application of ...
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Does immunity to CRISPR proteins limit their effectiveness?
The use of crisper-cas systems is currently applied to cells cultivated in vitro. As control of the ‘off target’ effects of Crispr improves and Crispr is used in vivo, why won’t the immune system ...
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Gene knockdown vs gene knockout vs knocksideways? [closed]
How are the techniques: Knock-sideways, knockout & knock-down different?
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Can CRISPR be used in editing primary cells? How is the transfection efficiency?
Sometimes people wanna use primary cells to do gene-editing because the cells normally have more interesting characteristics, but can we really do so?
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Does using CRISPR/Cas9 knockout need to add donor DNA in the process?
I am new to this technology and don't quite understand how it works. Hope someone can give some suggestions! Thank you.
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simple explanation of a crispr screen vs pooled crispr screen?
I'm familiar with how CRISPR works to make either knockdown or amplification effects for a single gene because you can make precision cuts.
What exactly does a pooled crispr screen do & how does ...
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CRISPR-Cas9 knockout
With CRISPR-Cas9 I have conducted a targeted knockout of a DNA region encoding a certain protein (working with leukocytes). My question is, how long does it take until this protein is not detectable ...
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What software do I need to read-write cas9? and .dna files?
I am still a complete beginner to crispr and I am still trying to learn what it is and how to actually use it. I now realise that you have to order the crispr components after you have actually ...
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How to design a permanent vaccine to cholera using CRISPR?
I know that Vibrio Cholerae infects the body through the GM1 ganglioside. So, would it be possible to engineer a CRISPR gene editing tool to prevent Vibrio Cholerae from getting into our cells?
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How to find the enhancer region of a specific gene?
I am new to these concepts in biology and need some help understanding. My main concern is how to find the enhancer of my gene of interest, specifically the sequence of the enhancer. I am working on a ...
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what's the difference between traditional genetic engineering and and CRISPR?
While at school, I learnt about restriction enzymes and how you could insert foreign DNA into an organism.
When I first heard about CRISPR I thought it was very similar except much more precise/...
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Can Cas13 be used with multiple crRNAs in the same reaction?
CRISPR-Cas13 equipped with crRNA (complementary to transcripts of interest) can be designed to target ssRNA transcripts in cells.
Upon successful crRNA and ssRNA binding, a fluorescent domain on ...
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CRISPR/Cas9 edited E.coli on AFM?
I am doing CRISPR/Cas9 experiment on E. coli. I am introducing recombinant plasmid BPK764 (which carries Cas9 + sgRNA designed and added later in that plasmid) into compentent E.coli cells which ...
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What is meant by "heads" and "tails" in the context of gene orientation?
I have a hard time understanding what this paper is talking about when it says:
We observed maximal cleavage at sites oriented tail-to-tail and separated by -10 bp to +30 bp (Fig. 2d). Finally, ...
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Can CRISPR also remove DNA viruses?
If I'm not mistaken only RNA viruses insert themselves into the host genome. As an example of DNA viruses, herpes viruses for example do not insert themselves in the host genome.
Can CRISPR cut DNA ...
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How do DNA viruses keep themselves in the nucleus without inserting themselves into genome?
If I'm not mistaken only RNA viruses insert themselves into the host genome. As an example of DNA viruses, herpes viruses for example do not insert themselves in the host genome.
Then how do DNA ...
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DIYbio - CRISPR injection sites for targeting the ABCC11 gene [closed]
I've been researching into the biohacking world where people most notable Josiah Zayner and Tristan Roberts have used a CRISPR solution developed in their backyard for gene therapy.
There is even a ...
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Size constraints on CRISPR guide RNA
I had a quick questions on the size limitations of a CRISPR guide. More specifically on the shorter end. Can I make a guide that is say 7-10bp and still have an active complex? I transfect using an ...
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When gene editing are both chromosomes in a pair changed?
Sorry for the possibly confused question, my knowledge of genetics is limited to medical training only but I have a question.
Are gene editing techniques such as CRISPR used on both of the ...
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Can CRISPR-cas9 be used to insert a large gene?
I would like to insert a 1500 bp or longer gene to the broken site in E.coli (or may be bacillus subtilis which support NHEJ) after cut with Crispr-Cas9. Is this possible for both NHEJ or HDR?
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Crispr complex in human cells?
Is the crispr (where the parts of Virus DNA is saved) section of the DNA existing in human cells aswell or is it just in bacteria cells?
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What is the role of CRISPR-dCas9 in gRNA-dCas9 transcription regulator complexes?
In this paper1, I read that mutant versions of Cas proteins such as a deactivated Cas9 (dCas9) are used alongside a guide-RNA (gRNA) to form variants of CRISPR tool that can function as transcription ...
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Mathematical models of gene editing using CRISPR
One confusing thing I have found when reading articles about possible CRISPR based gene therapy treatments in humans is that there is vey little discussion about what percentage of your cells will ...
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Does gene editing have any technical cross-species limits?
How far could you go in cross-species gene editing?
Is it possible for example to introduce plant genes into human DNA and vice versa?
Question: How is gene editing limited by the "donor" and "...
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How was Crisper Cas9 function discovered?
What paper outlines the discovery of how the CRIPER DNA, CrRNA, and Cas9 interact?
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Crispr/CAS9 genome editing is actually processed at which phase of Cell cycle?
all,
Is it only happens in certain phases, like S, G1,...or it can happen any time....or maybe, it has some perferences.
Put it in another word, does it going to processe edit if the cell is not ...
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How could a species be engineered to go extinct?
Non-biology background here.
I read this very interesting article: https://www.wired.com/story/crispr-eradicate-invasive-species/
However I am having a hard time wrapping my head around something:
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