Part:BBa_K2457004
Donor DNA for HDR into LacZ
This BioBrick is the key part of the CRISPeasy-in (crIN) framework, developed in 2017 by the Amazonas_Brazil team to make genome editing based on CRISPR/Cas9 easier and accessible for all.This construction consists of the template DNA gene that will be inserted in the edited genome. It is the first registry vector for a Donor DNA.
Figure 1: BBa K2457004 circuit
Usage and Biology
Composed by: the JK26 promoter, the coding sequence for the Green Fluorescent Protein (GFP) and the ilvBN terminator. It has 1810bp and was designed to contain two homologous arms with 300bp upstream and downstream from the Operon Lac region in the bacterial genome. Acording to Zhao et al (2016), the use of recombination arms with 300bp increases the efficiency in 100%. On the other hand, if you have a 150bp arm, the efficiency decreases to 63%.
Between the homologous arms, lies the GFP gene, flanked by NheI sites, allowing an RFC10 versatile one-step compatibility, the insertion of any other gene in GFP site.
It has this structure especially so the DSB repair will be guided to the HDR (Homology Direct Repair), where homology is recognized by the RecA enzyme, which will break through the BioBrick to restore the cleaved sequence in the genome, having the aimed gene integrated.
Design
Characterization
BBa_K2457000 was used by Amazonas_Brazil team in CROUT experiments and CRIN experiments.
CRIN (CRISPeasy-in method): Designed for knock-in strategy for bacterial genome editing based on CRISPR/Cas9</p>
RESULTADOS DE CRIN AQUI JU
Sequencing
Figure 2:Sequencing electropherogram from BBa_K2457004
Figure 3:Alignment of the designed sequence and our final construction from BBa_K2457004.
Sequence and Features</p>
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 321
Illegal NheI site found at 1164 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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