Half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, C-split Cas9 + DnaE C-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.
As the part needs to be inserted in a viral vector, the overhangs have to be designed accordingly to the viral vector that is going to be used. The synthesis of N-split Cas9 could not be made in one gBlock, as it is too big. Hence, the half protein had to be splitted in two other parts for its synthesis.
The protein domain is synthesized with the intein
Cas9 is an RNA-guided double-stranded DNA endonuclease, which belongs to the type II bacterial CRISPR system, from Streptococcus pyogenes. Dead cas9(dcas9), in which two mutations were introduced to abolish the endonuclease function, is added behind the biobrick J13002. Here we combine dcas9 with several different guide RNAs to activate or repress one of the double promoters??? transcription.
We made point mutation of EcoRI Restriction Enzyme cutting site inside the coding region of dcas9.
Dcas9 was provided by addgene. Cas9 is a gene from Streptococcus pyogenes.
The NEU-China iGEM team 2016 designed a fusion protein consisting of tCas9 and CIBN for sequence-specific transactivation of a desired target locus (more information). We used our double truncated tCas9 (BBa_K1982007) impaired in its cleavage activity and fused it to the CIBN (BBa_K1982003). An prokaryotic RBS sequence from the Community collection(BBa_B0034) fused to the beginning of tCas9-CIBN. For detection of expression the fusion protein was tagged with a HA-epitope coding sequence (BBa_K1150016) .Figure 1 illustrates the detailed design of the whole device. Figure 1: Construct design. tCas9 was fused to CIBN. The resulting fusion construct was flanked by RBS sequences and tagged by a HA epitope. The pBad/araC promoter and rrnB T1 terminator were chosen to control gene expression. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1]. we fused the light-inducible heterodimerizering proteins CRY2 and CIB1 from Arabidopsis thaliana to the VP64 transactivation domain and C-terminus of tCas9.
The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a tCas9 fused to its N terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription.
CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
dCas9 is a mutant of Cas9, with its endonuclease activity wiped out. dCas9 acts as the sole protein in CRISPRi system (Qi et al., 2013). Reference QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
Mutation: Aspartate 10 to Alanine (D10A) and Histidine 840 to Alanine (H840A) in order to cripple the endonuclease activity.
Promoter sequence containing multiple PAMs staggered at regular intervals to provide multiple possible sites for dCas9 binding.
tCas9 is a standardized (RFC 10) protein.Interacting with a DNA-binding RNA and fused with different effector domains it can be used for specific gene regulation. Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA.This recognition is based on Watson Crick base pairing between a short RNA, called crRNA, and the complementary DNA strand. A second RNA, called tracrRNA, connects crRNA and Cas9. These three parts together form a protein-RNA-DNA complex with the targeted DNA strand [1] Cas9 became of great interest for research concerning DNA targeting, because of its ability to recognize site specific DNA strands by a crRNA. At first the functionality of Cas9 was modified by exchanging aminoacids. As a result, Cas9 was able to introduce mutations within the genome of several organisms by causing double strand breaks [2][3]. Then, it was converted from a nuclease to a nickase introducing single strand breaks [4] and lately it was converted to an enzymatically inactive form, called dCas9 [5]. This tCas9 is standardized (RFC 10). It can be used as a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.
mutant Pst 1 :gctacctgcaG to gctacctgcaC mutant Pst 1 :actatctgcaG to actatctgcaC
GE Share [1] Westra E.R., Swarts D.C., Staals R.H., Jore M.M., Brouns S.J., van der Oost J. (2012). The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity. Annu Rev Genet. 46, 311-39 [2] Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121), 823-6 [3] Jiang W., Bikard D., Cox D., Zhang F., Marraffini L.A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 31(3), 233-9 [4] Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339 (6121), 819-23 [5] Qi L.S., Larson M.H., Gilbert L.A., Doudna J.A., Weissman J.S., Arkin A.P., Lim W.A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5), 1173-83
Synthetic vector pET-T7p(-12T)-SpacerA-GFP-LVA
Superfolder GFP. 488nm Excitation, 530nm Emission
35s:5' + TFL consense + SAGTI-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with SAGTI linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'.
This part was obtained using GoldenBraid assembly. The Ga20ox consense sequence was obtained with oligonucleotide synthesis.
sgRNA with 5' golden gate adapter and COM protein binding site
For use in CRISPR/Cas9 systems. Contains golden gate adapter at 5' end for inserting target sequence. Contains a COM protein binding site in the middle.
Part containing only dCas9 enzyme with no terminator or promoter. Interacts with sgRNA to bind to specific DNA target.
sgRNA with PAM-Proximal complementary sequence 5
Eukaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)
Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . This part is full-length CRY2 CDS. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1].
mutant EcoR 1 : TGTTGGGAATTc--->TGTTGGGAATTg
The light-inducible heterodimerizering proteins CRY2 and CIB1 are both from Arabidopsis thaliana.
The human codon optimized Cas9 from the organism Streptococcus pyogenes was standardized (RFC 25). Additionally we mutated the Nickase function. In the end we engineered a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.
Restriction sites were deleted by primer overhangs. Parts were fused together by fusion PCR. The nickase function was mutated in order to abbolish the demolition of DNA.
35s + Ga20ox consense + AEK-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with AEK linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'
sgRNA with dCas9 binding site sequence 9 and PP7 handle insert
For use in CRISPR/Cas9 systems. Contains golden gate adapter at 5' end for inserting target sequence. Also contains second golden gate adapter between truncated terminator and aspA terminator. Both golden gate adapters contain Bsa1 cut sites.
sgRNA with 5' golden gate adapter and MS2 coat protein binding site
For use in CRISPR/Cas9 systems. Contains golden gate adapter at 5' end for inserting target sequence. Contains a MS2 coat protein binding site in the middle.
This rather simple part contains the crRNA. The crRNA contains the target sequence to which the Cas9 protein is guided. The crRNA part can be cloned in front of any promoter. Note, that no RBS should be between the promoter and the crRNA. The crRNA is one of the two RNAs (crRNA and tracrRNA) that are needed to build the RNA dimer complex that fuses with the Cas9. The crRNA and the tracrRNA dimerize and are processed. This complex then binds to the CAS9. The RNA hybrid guides the Cas9 to its target and the Cas9 generates a site-specific double strand break. This crRNA here encodes for a sequence of the Kanamycin resistance gene cassette. The design of the crRNA is taken from the Jian et al. 2013 paper.
This part was synthesized by IDT. THe design was taken from the paper below: Wenyan Jiang, David Bikard, David Cox, Feng Zhang & Luciano A Marraffini (2013): RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31 (3), 233-239.
CRISPR/Cas9 system with pLac inducible promoter
coding sequence of Cas9 from CRISPR system type II
CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system provide protection against mobile genetic element by guided specific DNA cleavage. Cas9 (CRISPR-associated protein 9) belongs to type II CRISPR system and contains HNH motif and three RuvC-like motifs, homologous to HNH and RuvC endonuclease, respectively. Cas9 has strong DNA cleavage activity, thus it showed potential use in gene editing. This part contains the coding sequence of cas9 from Streptococcus pyogenes (UniProtKB AC number:Q99ZW2). Some "redundant" sequence was added to ensure the Cas9's normal function or just for the convenience to detect it in cell. The cas9 protein must be guided by two RNAs, crRNA(CRISPR RNA) and tracrRNA(trans-activating crRNA), to complete its specific DNA cleavage function. We build another part-a chimeic RNA-that combines the feature two RNAs. For more details, refer to part BBa_K1160001. With this part, you can 1)directly use it to express cas9 protein in cell or 2)use T7 RNA polymerase to get cas9's mRNA, which can be further modified to prepare authentic mRNA that can be used for translation in vivo.
35s:5'+ Ga20ox consense + SAGTI-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3') It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with SAGTI linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
Prokaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)
Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . This part is full-length CRY2 CDS. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" ! colspan="2" style="background:#FFBF00;"|VP64 |- |'''Function''' |transcription activator |- |'''RFC standard''' |[http://parts.igem.org/Help:Assembly_standard_10 RFC 10] |- |'''Backbone''' |pSB1C3 |- |'''Organism''' |''synthetic construct'' |- |'''Source''' |synthetic construct |- |'''Submitted by''' |[http://2016.igem.org/Team:NEU-China NEU-China 2016] |} Tetrameric VP16 transcription activator domain
Constitutively Expressed gRNA targeting mRFP
This Constitutively Expressed gRNA targeting mRFP is a no-BioBrick-scars assembly of the constitutive promoter BBa_J23100, DNA sequence of a gRNA that targets mRFP reporter and a reliable terminator BBa_B0015. This gRNA sequence comes from the following literature: QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
gRNA shall be exactly transcribed out. Any extra nucleotides would potentially jeopardize the normal function of the gRNA. Therefore, BioBrick scars are not allowed here. Instead of cut-and-ligation, inverse PCR or In-Fusion is preferred for the assembly task. In addition, a reliable terminator is applied here to ensure the termination of gRNA.
The gRNA sequence is de novo synthesized.
35s + TFL consense + AEK-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with AEK linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
This part was obtained using GoldenBraid assembly. The TFL consense sequence was obtained with oligonucleotide synthesis.
This part is submitted under the RFC 65 standard. Stay tuned for more description, the MIT 2013 Team.
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, which allow for the easy cloning of new spacers.
Mutagenesis to remove internal EcoRI site
sgRNA containing two golden gate adapters
Synthetic modification of crRNA and tracrRNA found in S. pyrogenes
To simplify the traditional gRNA comprised of a crRNA containing a dCas9 handle, and tracrRNA which binds to the PAM sequence in CRISPR, we created a joined sgRNA. As modifying the 3' end of the gRNA does not affect dCas9 binding we have used a deactivated dcas9 terminator and placed golden gate adaptable sites. The two sites are located where the complementary PAM-proximal sequence is located, allowing the distance dCas9 binds from the promoter to be adapted.The other golden gate adaptor in the position after the deactivated terminator so any RNA sequence such as an RNA binding protein handle can be inserted. This region is then followed by a real terminator for the strand.
tCas9 is from GE Share company. VP64 is synthetic construct.
35s + Ga20ox consense + RSIAT-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with RSIAT linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
This Constitutively Expressed dCas9 Operon is an assembly of the constitutive promoter BBa_J23100, a strong RBS BBa_B0034, coding sequence of dCas9 protein and a reliable terminator BBa_B0015. dCas9 is a mutant of Cas9, with its endonuclease activity wiped out. dCas9 acts as the sole protein in CRISPRi system (Qi et al., 2013). Reference QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
The expression level of dCas9 shall be delicately controlled, so as to acquire continuous adjustment (regulation) of target genes.
CRISPR/Cas9 system with Anderson high-expression constitutive promoter
Trevor Kalkus, Gordon Wade, Alissa Greenberg
The E. coli CRISPR system uses the CASCADE complex to target invasive DNA. CasA is the first subunit of the CASCADE complex and is responsible for the initial identification of invasive DNA. The ancestral version of this has been reconstructed using the seed sequences from the PFAM data base by creating a phylogenetic tree using PhyML and then using the WAG method in PAML with Lazarus. This part can be used with casBCDE to create the full CASCADE protein complex.
rearranged CRISPR/Cas9 system without promoter
spacer placeholder containing BsaI sites
Region containing bidirectional constitutive promoter
sgRNA with 5' golden gate adapter and PP7 protein binding site
For use in CRISPR/Cas9 systems. Contains golden gate adapter at 5' end for inserting target sequence. Contains a PP7 coat protein binding site in the middle.
RBS used for superfolder GFP in team Warwick 2016's project.
Contains outward facing Bsa1 cut sites for golden gate assembly of parts.
CRISPR/Cas9 system with pRha inducible promoter
This complex part contains the tracrRNA and the CAS9 under the control of constitutive promoters. Note that there is no RBS before the tracRNA. The tracrRNA-CAS9 part is the heart of the CRISPR system. The tracrRNA and the corresponding target crRNA dimerize and are processed and hybridize before binding to the CAS9 protein. The tracrRNA has no specific part that is responsible for binding to the targeted DNA. Therefor this part can be used together with any crRNA. The CAS9 gene encodes for the CRISPR associated protein 9 that is needed to generate a double strand break at a target site. CAS9 binds to the tracRNA-crRNA dimer/gRNA and is then guided by them/it to the target site where it generate a double strand break. This part can be used together with the crRNA anti KAN or the gRNA anti KAN and cause double strand breaks. It can of course also be used together with new generated crRNAs/gRNAs. The tracRNA-CAS9 part is taken from the CRISPER plasmid of the Jiang et al. 2013 paper.
The plasmid was ordered from Addgene and come originally from the paper below: Wenyan Jiang, David Bikard, David Cox, Feng Zhang & Luciano A Marraffini (2013): RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31 (3), 233-239.
CRISPR/Cas9 system with Anderson medium-expression constitutive promoter
Encodes the N-terminal fragment of transcription factor CIB1 (cryptochrome-interacting basic-helix-loop-helix). CIB1 interacts with CRY2 (cryptochrome 2) in a blue light-specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 positively regulates FT expression. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1].
This part comes from the paper Liu, Yuchen, Yayue Zeng, Li Liu, Chengle Zhuang, Xing Fu, Weiren Huang, and Zhiming Cai. "Synthesizing AND Gate Genetic Circuits Based on CRISPR-Cas9 for Identification of Bladder Cancer Cells." Nature Communications Nat Comms 5 (2014): 5393.
This part has a sgRNA targeting lacI. It also has 5' and 3' ribozyme which cleave the RNA molecule with self catalysis so that any sequence can be added to 5' or 3' to this part without preventing Cas9 binding and its activity.
Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1]. we fused the light-inducible heterodimerizering proteins CRY2 and CIB1 from Arabidopsis thaliana to the VP64 transactivation domain and C-terminus of tCas9.
35s + Ga20ox consense + RSIAT-TEV-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with RSIAT+TEV linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
The E. coli CRISPR system uses the CASCADE complex to target invasive DNA. CasA is the first subunit of the CASCADE complex and is responsible for the initial identification of invasive DNA. This part can be used with casBCDE to create the full CASCADE protein complex.
We had to perform mutagenesis twice to remove PstI cut sites.
The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a tCas9 fused to its N terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription. tCas9 can be tagged with transcriptional activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcription activation of downstream target genes. This tCas9-based activators is the case that tCas9 fused directly to a single transcriptional activator( VP64).
CRISPR/Cas9 system with Anderson low-expression constitutive promoter
plasmid from GE Share company. [1] Westra E.R., Swarts D.C., Staals R.H., Jore M.M., Brouns S.J., van der Oost J. (2012). The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity. Annu Rev Genet. 46, 311-39 [2] Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121), 823-6 [3] Jiang W., Bikard D., Cox D., Zhang F., Marraffini L.A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 31(3), 233-9 [4] Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339 (6121), 819-23 [5] Qi L.S., Larson M.H., Gilbert L.A., Doudna J.A., Weissman J.S., Arkin A.P., Lim W.A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5), 1173-83
RBS used for dCas9 enzyme in team Warwick 2016's project.
35s + TFL consense + RSIAT-TEV-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with RSIAT+TEV linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
Half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, N-split Cas9 + DnaE N-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.
As the part needs to be inserted in a viral vector, the overhangs have to be designed accordingly to the viral vector that is going to be used. The synthesis of C-split Cas9 could not be made in one gBlock, as it is too big. Hence, the half protein had to be splitted in two other parts for its synthesis.
The protein domain is synthesized with the intein.
35s + TFL consense + RSIAT-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with RSIAT linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
Cas9 (optimized for expression in E. coli)
This part code for cas9 originated from S. pyogenes SF 370 with codon optimized for expression in E. coli.
sgRNA with PAM-Proximal complementary sequence 6
The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a VP64 fused to its C terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription.
CRISPR/Cas9 system with pBAD inducible promoter
Conversion of the iGEM parts registry to SBOL2.1