You observed that the CRISPR knock-out of protein X either promotes or represses RNA polymerase II transcription, depending on the gene. Which experiments would you carry out to reveal the mechanism for this differential activity?
At 19:30 there is some discussion of in-frame vs. out-of-frame deletions when looking to inactivate a protein. My question is: how common is it that deleting four amino acids wouldn't inactivate the protein?
Hi Max, great question. It's actually important to check for a "true" knockout, because even large disruptions and/or frame shifts don't always eliminate function. Sometimes frame-shifts in an exon will be "skipped," restoring the frame. Truncated/mutated proteins may also have residual function, etc. Some useful recent discussion about this on Twitter: twitter.com/JohnDoench/status/1188905016071020544 There are several papers on this that you might also want to check out: * Frameshift indels introduced by genome editing can lead to in-frame exon skipping (journals.plos.org/plosone/article?id=10.1371/journal.pone.0178700) * CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion (genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1237-8) * mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay. (journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007105) * Biological plasticity rescues target activity in CRISPR knock outs (www.nature.com/articles/s41592-019-0614-5)
Thank you
thanks
You observed that the CRISPR knock-out of protein X either promotes or
represses RNA polymerase II transcription, depending on the gene. Which
experiments would you carry out to reveal the mechanism for this
differential activity?
nice
At 19:30 there is some discussion of in-frame vs. out-of-frame deletions when looking to inactivate a protein. My question is: how common is it that deleting four amino acids wouldn't inactivate the protein?
Hi Max, great question. It's actually important to check for a "true" knockout, because even large disruptions and/or frame shifts don't always eliminate function. Sometimes frame-shifts in an exon will be "skipped," restoring the frame. Truncated/mutated proteins may also have residual function, etc. Some useful recent discussion about this on Twitter: twitter.com/JohnDoench/status/1188905016071020544
There are several papers on this that you might also want to check out:
* Frameshift indels introduced by genome editing can lead to in-frame exon skipping
(journals.plos.org/plosone/article?id=10.1371/journal.pone.0178700)
* CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion
(genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1237-8)
* mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.
(journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007105)
* Biological plasticity rescues target activity in CRISPR knock outs
(www.nature.com/articles/s41592-019-0614-5)
@@innovativegenomicsinstitute Wow, this is a very comprehensive and thoughtful answer. Thank you!
Want to be able to zoom in video. Msg for youtube.
6:47
Really complicated topic brothers