Difference between revisions of "Part:BBa K1218011"
(→‎WHU-China Team Contribution)
 
(17 intermediate revisions by 6 users not shown)
Line 6: Line 6:
 
This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites. Digestion with BsaI allows for insertion of a new spacer, thus changing the sequence targeted by Cas9.  
 
This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites. Digestion with BsaI allows for insertion of a new spacer, thus changing the sequence targeted by Cas9.  
  
−
<!-- Add more about the biology of this part here -->
 
−
===Usage and Biology===
 
  
−
'''Group:''' CU-Boulder, 2014
+
===Amazonas_Brazil team improvement===
  
−
'''Author:''' Josephina Hendrix
+
Amazonas_Brazil team has designed news BioBricks parts (<html><a target="_blank" href="https://parts.igem.org/Part:BBa_K2457001">BBa_K2457001</a></html> AND <html><a target="_blank" href="https://parts.igem.org/Part:BBa_K2457002">BBa_K2457002</a></html>) aiming to implement bioengineering principles into CRISPR locus. Our proposal was to rationally design parts to be interchangeable and standardized, providing a BioBrick toolbox to attend multiple SynBio purposes. For that matter, Amazonas_Brazil team has:
  
−
'''Summary:''' CU-Boulder demonstrated that BBa_K1218011 can be targeted to a specific DNA sequence through the modification of its spacer region. The original spacer was replaced with one that targeted a neomycin resistance gene. The modified and unmodified plasmids were transformed into cells containing the targeted gene and the decrease in growth with the target sample demonstrates the ability of the spacer to target a specific sequence.
+
• Engineered abstracted CRISPR locus, building two standard BioBricks parts: the <html><a target="_blank" href="https://parts.igem.org/Part:BBa_K2457001">BBa_K2457001</a></html> - composed of Cas9 coding sequence + BBa_B0015 -  and <html><a target="_blank" href="https://parts.igem.org/Part:BBa_K2457002">BBa_K2457002</a></html> - composed of an optimized sgRNA device.
 +
 
 +
• Built BBa_K2457001 without regulatory modules to provide an approach to easily switch the promoter and RBS from Cas9 coding sequence through 3A Assembly.  
  
 +
•  Designed abstracted parts with ALL RFCs assemblies compatibility, paving the way to many engineering possibilities.
  
−
'''Documentation:'''
+
•  Constructed BBa_K2457002 composed of both crRNA protospacer sequence and tracrRNA structure with an optimized design.
−
A 30mer spacer sequence targeting the neomycin phosphotransferase gene was designed and substituted for the original spacer in BBa_K1218011. BW23115 E. coli with the neomycin phosphotransferase inserted into the genome were chemically transformed with the original and modified BBa_K1218011 to compare CRISPR-Cas9 specificity. Transformants were selected for on chloramphenicol.
+
 +
•  Modified the Shine-Dalgarno (AGGAGG) from the RBS at the end of Cas9 coding sequence to avoid ribosome recruitment on the wrong place of our synthetic BioBrick. It was necessary as the Cas9 is located in an operon complex in native CRISPR locus.
  
−
[[File:Boulder_Improvement_upon_pCas9_FIGURE_1.png|600px|thumb|none|Figure 1: Transformation results of neomycin resistant ''E. coli'' with Cas9 part having either A) non-targeting or B) targeting spacer sequence.]]
+
===Part improvement===
  
 +
Cambridge-JIC 2016 has submitted a new part (<html><a target="_blank" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2148013">BBa_K2148013</a></html>) consisting of cas9 codon-optimized for <i>Chlamydomonas reinhardtii</i> chloroplast chassis. This has been achieved through software developed at Saul Purton's lab at UCL. All illegal sites have been removed whilst maintaining the codon information and genetic A/T bias of the system.
  
−
There was a substantial decrease in growth between the non-targeting (1920 colonies) and the targeting sample (8 colonies) that must be accredited to the differences in spacer sequence. As can be seen in Figure 1B), there is growth in the targeting sample. Sequencing showed that all eight colonies had deleted the spacer region and one or both of the adjacent repeats.
+
The cas9 submitted additionally has a fusion tag to link reporter genes such as <html><a target="_blank" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2148007">fluorescent markers</a></html>.
 +
 +
For more information on the contruction of this part please refer to the <html><a target="_blank" href="https://parts.igem.org/Part:BBa_K2148013:Design">design page</a></html>.
  
−
'''Group:''' CU-Boulder, 2014
+
The aim behind this part improvement is to use CRISPR/CAS9 technology to accelerate homoplasmy in chloroplast transformation and overcome this important bottleneck in plastid engineering. 
  
−
'''Author:''' Josephina Hendrix
 
  
−
'''Summary:''' Part BBa_K1218011 was further improved by the addition of the M13 packaging signal (BBa_K1445000) to form the composite part BBa_K1445001. This allows the part to be packaged into M13 phage and delivered to bacteria through phage rather than a transformation method.
+
==Egypt-AFCM Team Improvement==
  
  
−
'''Documentation:'''
+
[http://2017.igem.org/Team:AFCM-Egypt# Egypt-AFCM Team] aimed to use cas9 for non-coding RNAs editing represented in regulation of circular RNA based circuit to knock-in hsa_circ_0000064 at [https://parts.igem.org/Part:BBa_K2217001# BBa_K2217001] into Hepatocellular carcinoma cells. To improve characterization of [https://parts.igem.org/Part:BBa_K1218011# BBa_K1218011], We designed a CRISPR-based circuit with HDR (homology-directed repair) template for knock-in, to improve characterization CMV enhancer, CMV promoter and T7 promoter at [BBa_K2217000 BBa_K2217006 BBa_K2217007] were ligated to the same circuit with cas9, while HDR was ligated on a separate plasmid to be transfected to HepG2 cells for circuit evaluation. herein, Gel electrophoresis bands were described to document Cas9 characterization, while culture plates were also documented to compare the activity of both non-coding RNA circuit and CRISPR circuit, characterization data could be found at [https://parts.igem.org/Part:BBa_K1218011:Experience# BBa_K1218011:Experience]. Information about Our Team results can be found at [http://2017.igem.org/Team:AFCM-Egypt/Results# Egypt-AFCM Team Results]
−
The M13 packaging signal was inserted upstream of the CRISPR-Cas9 part and submitted to the registry as part BBa_K1445001. This new part was packaged into phage and introduced via infection to conjugated neomycin resistant bW23115 E. coli. Sample was plated on chloramphenicol to select for cells infected with the phage delivering the phagemid with the Cas9 part and M13 origin of replication.
+
  
−
[[File:Boulder_Improvement_upon_pCas9_FIGURE_2.png|300px|thumb|none|Figure 2: Infection results of BBa_K1445001 on a pSB1C3 backbone, grown on LB agar with 170 ug/mL chloramphenicol.]]
 
  
 +
==WHU-China Team Contribution==
 +
 +
{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
 +
 +
<I>[https://2023.igem.wiki/whu-china# WHU-China 2023]</I>
 +
|width='60%' valign='top'|
 +
<html>
 +
<div id="WHU-China">
 +
</div>
 +
</html>
 +
===1. Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve high genome editing efficiency in <i>E. coli.</i>===
 +
<p><b>Group:</b> [https://2023.igem.wiki/whu-china# WHU-China 2023]</p>
 +
<p>A research article indicated that Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve high editing efficiency in <i>E. coli</i> MG1655. The author reported 100% genome editing efficiency from randomly selected colonies(Fig1).<sup>[1]</sup></p>
 +
 +
<html>
 +
<figure style="text-align:center;">
 +
<img src="https://static.igem.wiki/teams/4630/wiki/parts/16.png" width="100%">
 +
<figcaption><b>Figure 1:</b> poxb gene knockout with 513 bp deletion: lanes 1,2 and 8 were control group samples, and others were experimental group samples. The Colony PCR product of the edited poxb gene was 1008 bp, and the original was 1521 bp.</figcaption>
 +
</figure>
 +
</html>
 +
 +
<p>We conducted ladder experiments on the arabinose induction time to figure out the optimal duration of induced editing. As it is said that the induction should last at least for 6h<sup>[1]</sup>, ladder induction was set up from 6h to 30h. Although double bents always existed, which means that bacteria are not completely edited in this colony (Fig2), 24h was considered as the optimal induction time with minimal double bents and almost 100% editing efficiency (Fig3).</p>
 +
 +
<html>
 +
<figure style="text-align:center;">
 +
<img src="https://static.igem.wiki/teams/4630/wiki/parts/parts-13.webp" width="80%">
 +
<figcaption><b>Figure 2:</b> AGE results of genome editing</figcaption>
 +
</figure>
 +
<figure style="text-align:center;">
 +
<img src="https://static.igem.wiki/teams/4630/wiki/parts/parts-15.svg" width="80%">
 +
<figcaption><b>Figure 3:</b> Results of time ladder induction</figcaption>
 +
</figure>
 +
</html>
 +
 +
<p>Interestingly, we found that this system has higher genome editing rates in DH5-alpha than in MG1655. However, as it’s not the main part of our experiments and the lack of time, we didn’t make deeper investigations.</p>
 +
===2. Cas9 combined with Lambda-Red recombinases can achieve plasmid gene editing in <i>E. coli</i>.===
 +
<p>We confirmed that Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve plasmid gene editing. We added an N20s sequence and a batch of gRNAs targeting it into another plasmid (Fig4a). After co-transformation, we induced gene knockout for 24 hours by arabinose. It shows that many of the gRNAs (for example, NO. 11, 13, 14, 15) successfully targeted and deleted the N20s sequence (Fig4b).</p>
 +
 +
<html>
 +
<figure style="text-align:center;">
 +
<img src="https://static.igem.wiki/teams/4630/wiki/parts/parts-17.svg" width="100%">
 +
<figcaption><b>Figure 4a:</b> The design of plasmid N20s gene knockout</figcaption>
 +
</figure>
 +
<figure style="text-align:center;">
 +
<img src="https://static.igem.wiki/teams/4630/wiki/parts/parts-14.webp" width="100%">
 +
<figcaption><b>Figure 4b:</b> The results of plasmid N20s gene knockout</figcaption>
 +
</figure>
 +
</html>
 +
===3. It would be advisable to place Cas9 & Lambda-Red under an inducible promoter.===
 +
<p>The author of the research article informed us that constitutive gRNA expression has minimal impact on bacteria. However, it would be prudent to position Cas9 under an inducible promoter, such as araBAD. Cas9 can exhibit toxicity to bacteria, a phenomenon we observed in our experiments. When Cas9 is expressed through arabinose induction, the rate of culturing noticeably decreases compared to the previous conditions.</p>
 +
 +
===Reference===
 +
<p>[1] Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016).</p>
 +
|};
 +
 +
 +
<!-- Add more about the biology of this part here -->
 +
 +
===Usage and Biology===
  
−
The growth in figure 2 demonstrates that, with the addition of the M13 origin of replication, BBa_K1218011 can be delivered to cells via recombinant M13 phage.
 
  
 
<!-- -->
 
<!-- -->

Latest revision as of 19:26, 11 October 2023

Cas9

CRISPR-Cas is a bacterial immune system that remembers and targets foreign viral DNA by storing DNA sequences, or spacers, between clustered regularly interspaced short palindromic repeats (CRISPRs). RNA transcripts of the spacers are then used to sense homologous DNA, which is cleaved by CRISPR-associated (Cas) proteins.

This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites. Digestion with BsaI allows for insertion of a new spacer, thus changing the sequence targeted by Cas9.


Amazonas_Brazil team improvement

Amazonas_Brazil team has designed news BioBricks parts (BBa_K2457001 AND BBa_K2457002) aiming to implement bioengineering principles into CRISPR locus. Our proposal was to rationally design parts to be interchangeable and standardized, providing a BioBrick toolbox to attend multiple SynBio purposes. For that matter, Amazonas_Brazil team has:

• Engineered abstracted CRISPR locus, building two standard BioBricks parts: the BBa_K2457001 - composed of Cas9 coding sequence + BBa_B0015 - and BBa_K2457002 - composed of an optimized sgRNA device.

• Built BBa_K2457001 without regulatory modules to provide an approach to easily switch the promoter and RBS from Cas9 coding sequence through 3A Assembly.

• Designed abstracted parts with ALL RFCs assemblies compatibility, paving the way to many engineering possibilities.

• Constructed BBa_K2457002 composed of both crRNA protospacer sequence and tracrRNA structure with an optimized design.

• Modified the Shine-Dalgarno (AGGAGG) from the RBS at the end of Cas9 coding sequence to avoid ribosome recruitment on the wrong place of our synthetic BioBrick. It was necessary as the Cas9 is located in an operon complex in native CRISPR locus.

Part improvement

Cambridge-JIC 2016 has submitted a new part (BBa_K2148013) consisting of cas9 codon-optimized for Chlamydomonas reinhardtii chloroplast chassis. This has been achieved through software developed at Saul Purton's lab at UCL. All illegal sites have been removed whilst maintaining the codon information and genetic A/T bias of the system.

The cas9 submitted additionally has a fusion tag to link reporter genes such as fluorescent markers.

For more information on the contruction of this part please refer to the design page.

The aim behind this part improvement is to use CRISPR/CAS9 technology to accelerate homoplasmy in chloroplast transformation and overcome this important bottleneck in plastid engineering.


Egypt-AFCM Team Improvement

[http://2017.igem.org/Team:AFCM-Egypt# Egypt-AFCM Team] aimed to use cas9 for non-coding RNAs editing represented in regulation of circular RNA based circuit to knock-in hsa_circ_0000064 at BBa_K2217001 into Hepatocellular carcinoma cells. To improve characterization of BBa_K1218011, We designed a CRISPR-based circuit with HDR (homology-directed repair) template for knock-in, to improve characterization CMV enhancer, CMV promoter and T7 promoter at [BBa_K2217000 BBa_K2217006 BBa_K2217007] were ligated to the same circuit with cas9, while HDR was ligated on a separate plasmid to be transfected to HepG2 cells for circuit evaluation. herein, Gel electrophoresis bands were described to document Cas9 characterization, while culture plates were also documented to compare the activity of both non-coding RNA circuit and CRISPR circuit, characterization data could be found at BBa_K1218011:Experience. Information about Our Team results can be found at [http://2017.igem.org/Team:AFCM-Egypt/Results# Egypt-AFCM Team Results]


WHU-China Team Contribution

WHU-China 2023

1. Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve high genome editing efficiency in E. coli.

Group: WHU-China 2023

A research article indicated that Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve high editing efficiency in E. coli MG1655. The author reported 100% genome editing efficiency from randomly selected colonies(Fig1).[1]

Figure 1: poxb gene knockout with 513 bp deletion: lanes 1,2 and 8 were control group samples, and others were experimental group samples. The Colony PCR product of the edited poxb gene was 1008 bp, and the original was 1521 bp.

We conducted ladder experiments on the arabinose induction time to figure out the optimal duration of induced editing. As it is said that the induction should last at least for 6h[1], ladder induction was set up from 6h to 30h. Although double bents always existed, which means that bacteria are not completely edited in this colony (Fig2), 24h was considered as the optimal induction time with minimal double bents and almost 100% editing efficiency (Fig3).

Figure 2: AGE results of genome editing
Figure 3: Results of time ladder induction

Interestingly, we found that this system has higher genome editing rates in DH5-alpha than in MG1655. However, as it’s not the main part of our experiments and the lack of time, we didn’t make deeper investigations.

2. Cas9 combined with Lambda-Red recombinases can achieve plasmid gene editing in E. coli.

We confirmed that Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve plasmid gene editing. We added an N20s sequence and a batch of gRNAs targeting it into another plasmid (Fig4a). After co-transformation, we induced gene knockout for 24 hours by arabinose. It shows that many of the gRNAs (for example, NO. 11, 13, 14, 15) successfully targeted and deleted the N20s sequence (Fig4b).

Figure 4a: The design of plasmid N20s gene knockout
Figure 4b: The results of plasmid N20s gene knockout

3. It would be advisable to place Cas9 & Lambda-Red under an inducible promoter.

The author of the research article informed us that constitutive gRNA expression has minimal impact on bacteria. However, it would be prudent to position Cas9 under an inducible promoter, such as araBAD. Cas9 can exhibit toxicity to bacteria, a phenomenon we observed in our experiments. When Cas9 is expressed through arabinose induction, the rate of culturing noticeably decreases compared to the previous conditions.

Reference

[1] Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016).

;


Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1642
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3921
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4863
    Illegal BsaI.rc site found at 4840