Difference between revisions of "Part:BBa K5124011"

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This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type V CRISPR loci of <i>Lachnospiraceae bacterium ND2006</i> [1]. This sequence is combined with one of five spacer sequences that are complimentary to our target bovine TB DNA. Once transcribed into RNA, the 23-nucleotide repeat sequence folds into a single hairpin loop, which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA (Figure 1).  
 
This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type V CRISPR loci of <i>Lachnospiraceae bacterium ND2006</i> [1]. This sequence is combined with one of five spacer sequences that are complimentary to our target bovine TB DNA. Once transcribed into RNA, the 23-nucleotide repeat sequence folds into a single hairpin loop, which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA (Figure 1).  
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<img src = "https://https://static.igem.wiki/teams/5124/registry-images/cas12a-crrna.png" style = "height: 400px;aspect-ratio: auto 212 / 240">
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<i>Figure 1: Cas12a crRNA folded into the single hairpin loop</i>
  
 
===Characterisation===
 
===Characterisation===

Revision as of 14:15, 24 September 2024


Cas12a crRNA

Usage and Biology

The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems.

This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type V CRISPR loci of Lachnospiraceae bacterium ND2006 [1]. This sequence is combined with one of five spacer sequences that are complimentary to our target bovine TB DNA. Once transcribed into RNA, the 23-nucleotide repeat sequence folds into a single hairpin loop, which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA (Figure 1).

Figure 1: Cas12a crRNA folded into the single hairpin loop

Characterisation

This crRNA sequence was taken from the paper by Moreno-Mateos et al. [2]. It was synthesised by IDT as part of one of five composite parts (BBa_K5124030 to BBa_K5124034) each containing: a 3’ spacer sequence (BBa_K5124013 to BBa_K5124017), 5’ T7 promoter BBa_I719005 and BioBrick compatible prefix and suffixes. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker.

Please see composite parts:
K5124030
K5124031
K5124032
K5124033
K5124034
for further usage and results.

References

[1] Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22; 163(3):759-71.

[2] Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, et al. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun. 2024 Dec 8; 8:1-9.

[3] Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19(3):259-68.

Sequence and Features


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]