Difference between revisions of "Part:BBa K2130006"
Line 6: Line 6:
  
 
Cpf1 is an endonuclease that analogue to Cas9. To facilitate recognition of target sites by Cpf1, it needs a T-rich PAM sequence(TTTN) that is 5' of spacer.Since Cpf1 depend on different PAM sequence compared to Cas9, it increases the possibilities to target new genes. Lastly, Cpf1 endonuclease creates a staggered/ stick-end DNA double stranded breaks instead of blunt-end breaks.  
 
Cpf1 is an endonuclease that analogue to Cas9. To facilitate recognition of target sites by Cpf1, it needs a T-rich PAM sequence(TTTN) that is 5' of spacer.Since Cpf1 depend on different PAM sequence compared to Cas9, it increases the possibilities to target new genes. Lastly, Cpf1 endonuclease creates a staggered/ stick-end DNA double stranded breaks instead of blunt-end breaks.  
 +
 +
As shown in the results below, LbCpf1 needs to be guided by gRNA that range from 19-23 nucleotides in order to perform optimally for both NHEJ and HDR repair mechanisms. Additionally, it is interesting to find that Cpf1 achieves a higher HDR efficiency compared to Cas9 endonuclease.
  
 
<html><center><img src="https://static.igem.org/mediawiki/parts/8/86/LBCpf1.PNG" height="60%"width="75%">
 
<html><center><img src="https://static.igem.org/mediawiki/parts/8/86/LBCpf1.PNG" height="60%"width="75%">

Revision as of 12:39, 24 October 2016


LbCpf1

Human codon optimised Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1)

Cpf1 is an endonuclease that analogue to Cas9. To facilitate recognition of target sites by Cpf1, it needs a T-rich PAM sequence(TTTN) that is 5' of spacer.Since Cpf1 depend on different PAM sequence compared to Cas9, it increases the possibilities to target new genes. Lastly, Cpf1 endonuclease creates a staggered/ stick-end DNA double stranded breaks instead of blunt-end breaks.

As shown in the results below, LbCpf1 needs to be guided by gRNA that range from 19-23 nucleotides in order to perform optimally for both NHEJ and HDR repair mechanisms. Additionally, it is interesting to find that Cpf1 achieves a higher HDR efficiency compared to Cas9 endonuclease.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 25
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 965
    Illegal BglII site found at 1298
    Illegal BglII site found at 1868
    Illegal BglII site found at 2135
    Illegal BglII site found at 2756
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1356
    Illegal SapI.rc site found at 1416