Difference between revisions of "Part:BBa K4727109:Design"

 
(→‎Design Notes)
 
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===Design Notes===
 
===Design Notes===
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The sequence has been designed considering the predicted precision of the guide and the possible off targets
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The sgRNA is composed of a 3' constant region, the scaffold, and a 5' variable region, corresponding to 17-24 nt complementary to the target DNA.
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Focusing on the scaffold, we have found that the same 83 nt long sequence (developed by Qi et al., 2013), including the handle for the dCas9 and a terminator from S. pyogenes, was used in all the strains of interest during different studies.
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+
One of the most critical difficulties in using CRISPR/Cas, and thus CRISPRi, is the design of a specific sgRNA to prevent a high number of off-target sites. Cui et al (2018) reviewed a list of tools for on and off-target predictions in the proper design of sgRNA, such as CHOPCHOP (https://chopchop.cbu.uib.no/) and CasOFFinder (http://www.rgenome.net/cas-offinder/) respectively. It is important that there are no single base mutations and therefore no mismatches with the target DNA in the first 12-7 nt at 3' end (seed region) of the guide; otherwise, the effectiveness and specificity of the system decrease
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In order to insert the sgRNA sequences into the plasmids used for the bacteria, we decided to synthetize fragments of DNA consisting of the entire expression cassette for the sgRNAs, flanked by the Prefix and the Suffix.
  
 
===Source===
 
===Source===

Latest revision as of 08:42, 23 September 2023


sgDNA OmpA 157 for A. baumannii


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The sgRNA is composed of a 3' constant region, the scaffold, and a 5' variable region, corresponding to 17-24 nt complementary to the target DNA. Focusing on the scaffold, we have found that the same 83 nt long sequence (developed by Qi et al., 2013), including the handle for the dCas9 and a terminator from S. pyogenes, was used in all the strains of interest during different studies. One of the most critical difficulties in using CRISPR/Cas, and thus CRISPRi, is the design of a specific sgRNA to prevent a high number of off-target sites. Cui et al (2018) reviewed a list of tools for on and off-target predictions in the proper design of sgRNA, such as CHOPCHOP (https://chopchop.cbu.uib.no/) and CasOFFinder (http://www.rgenome.net/cas-offinder/) respectively. It is important that there are no single base mutations and therefore no mismatches with the target DNA in the first 12-7 nt at 3' end (seed region) of the guide; otherwise, the effectiveness and specificity of the system decrease In order to insert the sgRNA sequences into the plasmids used for the bacteria, we decided to synthetize fragments of DNA consisting of the entire expression cassette for the sgRNAs, flanked by the Prefix and the Suffix.

Source

This sequence has been designed with bioinformatic tools based on the analysis of the genomic sequence of Acinetobacter baumannii.

References