Difference between revisions of "Part:BBa K5124011"

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<partinfo>BBa_K5124011 short</partinfo>
 
<partinfo>BBa_K5124011 short</partinfo>
 
===Usage and Biology===
 
===Usage and Biology===
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The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems.
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The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.
  
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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).  
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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> [3]. 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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===Characterisation===
 
===Characterisation===
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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_K5124030 to BBa_K5124034) each containing: a 3’ spacer sequence (BBa_K5124013 to BBa_K5124017), 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.
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This crRNA sequence was taken from the paper by Moreno-Mateos <i>et al.</i> [4]. 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 [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 [5]) carrying an ampicillin selection marker.
  
 
Please see composite parts:   
 
Please see composite parts:   
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===References===
 
===References===
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[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.  
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1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.
  
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[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.  
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2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.
  
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[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.  
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3. 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.
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4. 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.
 +
 
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5. 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===
 
===Sequence and Features===
 
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Revision as of 21:33, 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. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.

This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type V CRISPR loci of Lachnospiraceae bacterium ND2006 [3]. 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. [4]. 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 [5]) carrying an ampicillin selection marker.

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

References

1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.

2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.

3. 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.

4. 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.

5. 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]