Hey guys I'm trying to knock out a gene in a fast growing species of mycobacterium called M.smegmatis. The techniques I came across involve one and two step recombination techniques with pGOAL and pNIL plasmids. I'm completely new to all but the simplest genetic modification so this will be a stab in the dark. I was wondering would another possibility be the CRISPR technique? Does it work in bacterium?
Best wishes
Harry
CRISPR technique can be used if you have the genome of your organism sequenced. You simply need to design the sgRNA for your gene of interest and follow the normal protocols.
Even if you know the sequence of only your gene of interest, you can still do this by searching for bioinformatic tools that can do this type of job.
CRISPR/Cas9 mediate GE in bacteria is possible and widely reported . Here is one of the detailed paper published in 2013 that might help you.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748948/pdf/nihms436755.pdf
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Hi Harry
Many useful references on attachment 1
https://nar.oxfordjournals.org/content/early/2016/07/12/nar.gkw625.full
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https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0595-3
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https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0ahUKEwj7yLvPoqnUAhXIPRoKHUF8AAkQFghZMAU&url=https%3A%2F%2Fmed.stanford.edu%2Fqilab%2Fpublications%2F_jcr_content%2Fmain%2Fpanel_builder_19%2Fpanel_0%2Fdownload%2Ffile.res%2FPeters_Curr_Microbiol_2015.pdf&usg=AFQjCNGw44cGmZqCsrOvRHHX8qMfVteglQ&sig2=ZB2USvMRJVF4KkAHgUCDCQ
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https://academic.oup.com/nar/article/44/9/4243/2462317/Consequences-of-Cas9-cleavage-in-the-chromosome-of
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https://www.semanticscholar.org/paper/Programmable-transcriptional-repression-in-mycobac-Rock-Hopkins/16a94ba6bbec19db20f79834e7880d5678fb966e
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https://www.scbt.com/scbt/browse/gene-editing-CRISPR-Knockout-Activation-Products-lymphocyte-signaling-crispr-knockout-and-activation-products-granulysin-CRISPR-Plasmids/_/N-tijd07
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https://dpb.carnegiescience.edu/sites/dpb.carnegiescience.edu/files/Bhaya_ARG.pdf
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https://journal.frontiersin.org/article/10.3389/fmicb.2015.00812/full
Hi there, whats the difference between GMO food (genetically modified organism) and gene edited (CRISPR) food ? Mutation occurs for both of them? And if a food is produced by CRISPR technique, is it also genetically modified? whats the exact meaning of editing? thank you in advance!
@ Sebahat Özakça,
I have posted a couples articles discussing similar question you asked on one of my projects (link below):
https://www.researchgate.net/project/collaboration-Working-on-a-scientific-manuscript--about-NBT-New-Breeding-Technologies-for-crop-improvement
@ Sebahat Özakça,
For your question: "Mutation occurs for both of them?"
It depends on your experimental design, both can cause mutation. For example, T-DNA (produce GMO) can insert into gene X, and cause gene X to mutate. CRISPR/sgRNA can also target to gene X and cause gene X to mutate (by causing DNA break inside the gene).
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