Is knockout by crispr and re-expression possible?

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Brian Reilly

I think it would be possible to "Rescue" the cells by transducing them with a lentivirus expressing your protein of interest. This would depend on the size of the gene you were trying to insert, and also likely on the cell type you are studying. I don't have much experience with this technique, but I know it has been used in papers like this one: http://www.nature.com/ng/journal/v47/n5/abs/ng.3258.html

Antonio Lázaro

Hi.

I have a problem with my research. I would like to study the function of one mutation. For that, I would like to remove the endogenous gene by CRISPR and then introduce the mutated gene by retroviral infection. But when I knocked out the gene in HEK293 by CRISPR, my cells died.

Have anyone tried to introduce first some gene and then knock out the endogenous gene using gRNA with homology in the introns? Do you think that it is possible?

Saikolappan Sankaralinga

yes you can transiently express your gene of interest after crispr knock out using a expression vector with CMV promoter like pcDNA3.

Yang Yu

This two-step approach can be used to study the function of a specific gene by first disabling it (knocking it out) and then restoring its expression in a controlled manner. Here's how the process typically works:

Knockout (Gene Disruption):CRISPR-Cas9 is used to target and edit the gene of interest in cells or organisms. This usually involves introducing a guide RNA (gRNA) that directs the Cas9 enzyme to cut the gene at a specific location. The Cas9-induced double-strand breaks in the gene trigger the cell's DNA repair mechanisms, which can result in insertions or deletions (indels) at the cut site. These indels can disrupt the gene's function, leading to a loss of gene expression or a non-functional gene product. This is the knockout step.

Re-expression:After achieving the knockout, the next step is to reintroduce the gene in a controlled manner to study its effects. This can be done by inserting the gene into the target cells or organisms using a suitable vector or delivery method. The gene can be placed under the control of a promoter that allows for regulated expression. Researchers can then assess the effects of re-expressing the gene in the knockout background and compare them to wild-type or control cells/organisms.

This approach allows scientists to investigate the function of a specific gene by first eliminating its expression (knockout) and then reintroducing it under controlled conditions (re-expression). It helps elucidate the role of the gene in various biological processes.

It's important to note that the success of both the knockout and re-expression steps can vary depending on the specific gene, cell type, delivery method, and experimental design. Additionally, the choice of promoter and regulatory elements for re-expression can influence the timing and level of gene expression.

l This list of protocols might help us better address the issue.