Composite
Kise Ryota

Part:BBa_K2200006:Design

Designed by: Meredith Eating Xiang   Group: iGEM17_Shenzhen_SFLS   (2017-07-22)


pU6+sgRNA+pHEf1A+hCas9


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 5117
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 863
    Illegal XhoI site found at 1262
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 997
    Illegal NgoMIV site found at 4214
    Illegal NgoMIV site found at 5123
    Illegal AgeI site found at 375
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 5242
    Illegal SapI.rc site found at 2633
    Illegal SapI.rc site found at 2875


Design Notes

U6 promoter drives the expression of sgRNA.

crRNA (CRISPR RNA)—contains both the 20 base protospacer element and additional nucleotides which are complementary to the tracrRNA. tracrRNA (transactivating RNA)—hybridizes to the crRNA and binds to the CAS9 protein activating the complex to creating double-stranded breaks at specific sites within genomic sequence. sgRNA (single-guide RNA)—combines the tracrRNA and crRNA, which are separate molecules in the native CRISPR/Cas9 system in S. pyogenes, into a single RNA construct, simplifying the components needed to use CRISPR/Cas9 for genome editing (for plasmid or IVT expression). A linker loop sequence is included between the two. PAM (protospacer adjacent motif)—a short sequence in either strand of the genome recognized by CRISPR nucleases as a cutting site. The recognized sequence will vary with the nuclease (e.g., Cas9, Cpf1, etc.). The protospacer sequence is comprised of the 20 bases directly upstream of the PAM site. protospacer element—the portion of the crRNA (or sgRNA) that is complementary to the genomic DNA target sequence; usually 20 nucleotides in length. RNA trigger—a generic term for the RNAs that activate the CRIPSR/CAS9 complex. They can be sgRNA or crRNA/tracrRNA.

Design of sgRNA: (a) The sgRNA is a chimera and consists of three regions: a 20–25-nt-long base-pairing region for specific DNA binding, a 42-nt-long dCas9 handle hairpin for Cas9 protein binding and a 40-nt-long transcription terminator hairpin derived from S. pyogenes. Transcription of sgRNAs should start precisely at its 5′ end. The 12-nt seed region is shaded in orange. (b) The schemes for designing sgRNAs to target the template (T) or nontemplate (NT) DNA strands. When targeting the template DNA strand, the base-pairing region of the sgRNA has the same sequence identity as the transcribed sequence. When targeting the nontemplate DNA strand, the base-pairing region of the sgRNA is the reverse-complement of the transcribed sequence.


Source

pHS-ACR-ZQ170

References

http://www.nature.com/nprot/journal/v8/n11/fig_tab/nprot.2013.132_F4.html