Difference between revisions of "Part:BBa K2130006"

Latest revision as of 12:44, 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. Another major difference between Cas9 and Cpf1 is that 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. Interestingly, Cpf1 is found to achieve 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]
Illegal NgoMIV site found at 1027
Illegal NgoMIV site found at 2026
Illegal NgoMIV site found at 2728
Illegal NgoMIV site found at 3717
Illegal NgoMIV site found at 3736
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1356
Illegal SapI.rc site found at 1416

@@ Line 5: / Line 5: @@
 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.
+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. Another major difference between Cas9 and Cpf1 is that 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. Interestingly, Cpf1 is found to achieve 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%">
 <img src="https://static.igem.org/mediawiki/2016/f/fc/T--NTU-Singapore--bigtest.jpg" height="60%"width="75%"></center>
 <img src="https://static.igem.org/mediawiki/2016/e/e2/T--NTU-Singapore--CDStest.jpg" height="60%"width="75%"></center>