During our journey into the “CRISPR world”, we have successfully designed and characterized several basic and composite parts as well as functional biological devices. Our parts portfolio contains 21 basic parts, 11 composite parts and 9 genetic devices. We encourage the synthetic biology community to “wander” around our genetic parts described below.
Best Basic Part
crRNA targeting the miR-17-3P BBa_K4170023
As our best basic part, we selected the crRNA targeting the miR-17-3P BBa_K4170023. This part contains the sequences of the stem loop (repeat part) and the spacer (detection part) required for the formation of the CRISRPR/Cas13a complex and the hybridization with the mature miR-17-3P, respectively. The LbuCas13a protein adopts a bilobed structure which consists of a a-helical REC lobe and a NUC lobe. The crRNA stem loop sequence is anchored to the space between the NTD and Helical-1 domain forming extensive contacts between the crRNA and the LbuCas13a protein. The crRNA spacer region (guide) is complementary to the miR-17-3P.
This part was inserted into pSB1C3 backbone BBa_K4170019 utilizing the methodology described on crPrep crRNA preparation. This method enables the standardized and time-saving reproducible design of any crRNA as well as the efficient insertion of any crRNA sequence into pSB1C3 plasmid backbone ensuring RFC [10] compatibility. For the precise design of the crRNA sequence, we relied on model-driven studying of the 3D-structure of the molecule as well as on the protein-crRNA docking results which are mentioned in detail on the registry. In addition, the detailed results of our cloning method as well as the in vitro transcription and purification process of the crRNA are also provided on the registry page.
Best Composite Part
SUMO-LbuCas13a coding device under T7 promoter (BBa_K4170016)
As our best composite part, we selected the SUMO-LbuCas13a coding device under T7 promoter BBa_K4170016. It constitutes an expression system of codon-optimized LbuCas13a protein in fusion with the small ubiquitin-like modifier (SUMO) protein, regulated by T7 promoter. This composite part contains the coding sequence (CDS) of the SUMO-LbuCas13a fused with 6XHis purification tag, and enables the recombinant SUMO-LbuCas13a protein expression in E.coli in fusion with the 6xHis affinity tag. The combined expression of LbuCas13a with SUMO protein, known as SUMO-fusion technology, improves the solubility of the recombinant protein LbuCas13a simplifying and facilitating purification and detection of the protein.
This plasmid has been constructed from pGJK_His-SUMO-LbuCas13a plasmid, deposited by Jennifer Doudna and her colleagues at Addgene plasmid repository, after successive mutagenesis PCR and cloning steps to remove the restrictions sites that are incompatible with RFC[10] biobrick standards. The final SUMO-LbuCas13a coding sequence has been successfully assembled with pSB1C3 backbone and lacks the 5 initial restriction sites (Enzyme: position in plasmid, XbaI:5030, SapI: 6263, EcorI:6987 & 8263, BsaI: 8339), ensuring the biobrick RFC [10] compatibility.
We recommend this part as a standardized system for the expression and purification of the SUMO-LbuCas13a protein which could be used by every iGEM's community team to facilitate the implementation of other synthetic biology projects. Having as an ultimate goal to motivate the subsequent teams to include the CRISPR/Cas13a system in their project, we have extensively characterized this system, as displayed in the registry, with the following:
- Providing information about the biology and usage of CRISPR/Cas13a systems.
- Specifying our cloning strategy.
- Investigating the conditions of the protein expression and highlighting the optimum conditions for efficient LbuCas13a production in the soluble cytoplasmic fraction. Our gel electrophoresis and western blot analysis results are also described on the registry.
- Scrutinizing the feasibility of CRISPR/Cas13a system as a miRNA detection tool, the buffer-related functionality of Cas13a, the receptor concentrations.
- Giving detailed information about the conditions for the conduction of the experiments enhancing the detection system's performance.
Parts in Registry
LbuCa13a collection
This collection contains all the LbuCa13a- related basic and composite parts that we efficiently designed and cloned into the pSB1C3 plasmid backbone. These parts are the necessary genetic building blocks for the construction of complex genetic devices for the LbuCas13a expression and purification. These parts can be used by future iGEM teams for the LbuCas13a expression as a single protein or in a fused form with SUMO tag which enhances the solubility of the recombinant protein. In addition, specific 6xHis affinity purification tags are added on the N-terminus of the LbuCas13a protein facilitating the purification step with ΗisTrap purification columns. The LbuCas13a/SUMO-LbuCas13a CDS can be transcribed under T7 (BBa_K4170017 / BBa_K4170016) or under rhamnose inducible rhaB promoter (BBa_K4170055) depending on the downstream applications.
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K4170020 | Coding | LbuCas13a codon optimized for E.coli | 3480 bp |
| BBa_K4170006 | Coding | LbuCas13a fragment A (1-849bp) | 848 bp |
| BBa_K4170003 | Coding | Lbucas13a assembly part 2 | 734 bp |
| BBa_K4170004 | Coding | Lbucas13a assembly part 3 | 1338 bp |
| BBa_K4170005 | Coding | Lbucas13a assembly part 4 | 646 bp |
| BBa_K4170006 | Coding | Lbucas13a fragment A (1-849bp) | 848 bp |
| BBa_K4170001 | Coding | bdSUMO solubility tag | 297 bp |
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K4170013 | Coding | LbuCas13a with 6XHis purification tag | 3543 bp |
| BBa_K4170014 | Coding | SUMO-LbuCas13a with 6XHis purification tag | 3840 bp |
| BBa_K4170016 | Device | SUMO-LbuCas13a coding device under T7 promoter | 5404 bp |
| BBa_K4170017 | Device | LbuCas13a coding device under T7 promoter | 5107 bp |
| BBa_K4170055 | Device | SUMO-LbuCas13a coding device under rhaB promoter | 5897 bp |
| BBa_K4170002 | Coding | bdSUMO- Lbucas13a assembly part 1 | 1240 bp |
| BBa_K4170008 | Coding | Lbucas13a assembly part 1 | 943 bp |
crRNA collection
This collection contains the crRNA sequences for the efficient CRISPR/LbuCas13a- based detection of the miR-17-3P and miR-17-5P. The crRNA sequences are located downstream of the T7 promoter for the in vitro transcription. In addition, the SapI restriction enzyme recognition site has been located at the end of the crRNA sequence to succeed the plasmid linearization which is required to terminate the T7 polymerase transcriptional process at the specified location. We chose the SapI enzyme since it is one of the most widely used type II S restriction enzymes in the synthetic biology community.
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K4170023 | RNA | crRNA targeting the miR-17-3P (standard design) | 50 bp |
| BBa_K4170024 | RNA | crRNA targeting the miR-17-3P (mismatch design) | 50 bp |
| BBa_K4170026 | RNA | crRNA targeting the miR-17-3P (extra loop design) | 55 bp |
| BBa_K4170027 | RNA | crRNA targeting the miR-17-5P (standard design) | 50 bp |
| BBa_K4170028 | RNA | crRNA targeting the miR-17-5P (mismatch design) | 55 bp |
| BBa_K4170029 | RNA | crRNA targeting the miR-17-5P (extra loop design) | 55 bp |
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K4170019 | Coding | crRNA targeting the miR-17-3P (standard design) under T7 promoter | 77 bp |
| BBa_K4170021 | Coding | crRNA targeting the miR-17-3P (mismatch design) under T7 promoter | 77 bp |
| BBa_K4170022 | Coding | crRNA targeting the miR-17-3P (extra loop design) under T7 promoter | 82 bp |
| BBa_K4170042 | Coding | crRNA targeting the miR-17-5P (standard design) under T7 promoter | 77 bp |
| BBa_K4170043 | Coding | crRNA targeting the miR-17-5P (mismatch design) under T7 promoter | 77 bp |
| BBa_K4170044 | Coding | crRNA targeting the miR-17-5P (extra loop design) under T7 promoter | 82 bp |
LbuCas13a-crRNA combined collection
This collection contains only composite parts combining the crRNA sequences from the crRNA collection with the LbuCas13a CDS from the LbuCas13a collection. The crRNA stability is significantly enhanced when it binds to Cas13a enzyme forming the Cas13-crRNA complex, thus we provide the iGEM community with the genetic devices required for the SUMO-LbuCas13a/crRNA simultaneous expression under T7 promoter in E.coli strains.
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K4170045 | Device | SUMO-LbuCas13a and crRNA targeting the miR-17-3P (standard design) coexpression system (all-in-one) | 5489 bp |
| BBa_K4170046 | Device | SUMO-LbuCas13a and crRNA targeting the miR-17-3P (mismatch design) coexpression system (all-in-one) | 5489 bp |
| BBa_K4170047 | Device | SUMO-LbuCas13a and crRNA targeting the miR-17-3P (extra loop design) coexpression system (all-in-one) | 5494 bp |
| BBa_K4170048 | Device | SUMO-LbuCas13a and crRNA targeting the miR-17-5P (standard design) coexpression system (all-in-one) | 5489 bp |
| BBa_K4170049 | Device | SUMO-LbuCas13a and crRNA targeting the miR-17-5P (mismatch design) coexpression system (all-in-one) | 5489 bp |
| BBa_K4170050 | Device | SUMO-LbuCas13a and crRNA targeting the miR-17-5P (extra loop design) coexpression system (all-in-one) | 5494 bp |
Regulatory collection
This collection contains basic and composite parts of regulatory genetic elements which are incorporated into our complex genetic devices. Specifically, this collection contains promoters with divergent regulatory control (T7 & pRha), the CDS of transcriptional regulators suitable for the transcriptional regulatory systems (lac repressor, RhaS & RhaR regulators), ribosome binding sites (RBSs), transcription terminators, etc.
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K4170030 | Regulatory | T7 promoter | 19 bp |
| BBa_K4170090 | Regulatory | lacI promoter | 78 bp |
| BBa_K4170091 | Coding | lac Repressor | 1083 bp |
| BBa_K4170007 | Other | Thrombin recognition and cleavage site | 18 bp |
| BBa_K4170009 | Conjugation | Serine-serine-glycine flexible linker | 9 bp |
| BBa_K4170012 | Other | methionine fused with GSS flexible linker | 12 bp |
| BBa_K4170051 | Coding | rhaR native derived from E.coli | 849 bp |
| BBa_K4170052 | Coding | rhaS native derived from E.coli | 837 bp |
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K4170054 | Composite | rhaBAD operon | 2031 bp |