Difference between revisions of "Part:BBa K5124011"

Revision as of 13:06, 22 August 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).

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_K5124035 to BBa_K5124040) each containing: a 3’ spacer sequence (BBa_K5124018 to BBa_K5124023), 5’ T7 promoter (https://parts.igem.org/Part:BBa_I719005 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: (https://parts.igem.org/Part:BBa_K5124035 K5124035) (https://parts.igem.org/Part:BBa_K5124036 K5124036) (https://parts.igem.org/Part:BBa_K5124037 K5124037) (https://parts.igem.org/Part:BBa_K5124038 K5124038) (https://parts.igem.org/Part:BBa_K5124039 K5124039) (https://parts.igem.org/Part:BBa_K5124040 K5124040) 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]

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K5124011 short</partinfo>
+===Usage and Biology===
+The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems.
-Common to all Cas12a crRNA sequences to put with the spacer
+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).
-<!-- Add more about the biology of this part here
+===Characterisation===
-===Usage and Biology===
+This crRNA sequence was taken from the paper by Moreno-Mateos <i>et al.</i> [2]. It was synthesised by IDT as part of one of five composite parts (BBa_K5124035 to BBa_K5124040) each containing: a 3’ spacer sequence (BBa_K5124018 to BBa_K5124023), 5’ T7 promoter (https://parts.igem.org/Part:BBa_I719005 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:
+(https://parts.igem.org/Part:BBa_K5124035 K5124035)
+(https://parts.igem.org/Part:BBa_K5124036 K5124036)
+(https://parts.igem.org/Part:BBa_K5124037 K5124037)
+(https://parts.igem.org/Part:BBa_K5124038 K5124038)
+(https://parts.igem.org/Part:BBa_K5124039 K5124039)
+(https://parts.igem.org/Part:BBa_K5124040 K5124040)
+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===
 <!-- -->
-<span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K5124011 SequenceAndFeatures</partinfo>