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How homologous do (endogenous) CRISPR array tracers need to be to degrade foreig

Crispr transformation genetic barriers

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#1 Chris22

Chris22

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Posted 03 February 2016 - 08:37 PM

Hello,

 

I am working against a series of genetic barriers to transformation in a bacteria which has never been successfully transformed

 

The genome shows the presence of an endogenous Type-II Crispr system which has an array of 14 spacers.

 

If I align these spacers with my plasmid of interest there is some pretty high levels of homology, not exact, but sometimes 100% identity across 60% of the spacer

 

 

Does anyone know how specific does the spacer/foreign sequence match have to be to be degraded by the endonuclease domain?

 

For example, if there is a few interspersed single bp differences, will this still be degraded? (like a primer/template relationship in PCR)

 

 

Also has anyone successfully used a crispr inhibiting protein (gene from phage) cloned to a plasmid to overcome a Crispr system? 

 

Thanks in advance

 

 







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