Part:BBa_K1982001

Prokaryotic tCAS9

tCas9
Function	Binding protein
RFC standard	RFC 10
Backbone	pSB1C3
Organism	Streptococcus pyogenes
Source	GE Share company
Submitted by	[http://2016.igem.org/Team:NEU-China NEU-China 2016]

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.

Usage and Biology

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)  ATGGACAAG ... GACGACAAATAATAA  ORF from nucleotide position 1 to 4344 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  301  401  501  601  701  801  901  1001  1101  1201  1301  1401  MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLHEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLHNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLARAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVGRADALDDFDLDMLGSDALDDFDLD MLGSDALDDFDLDMLGSDALDDFDLDMLINYPYDVPDYASDYKDDDDK*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) RFC25 scar (shown in bold):  657 to 658, 1102 to 1103 SV40 nuclear localization sequence:  1369 to 1375 HA-tag:  1431 to 1439 Flag-tag:  1441 to 1448 Enterokinase cleavage site:  1444 to 1448
Amino acid composition: Ala (A) 80 (5.5%) Arg (R) 80 (5.5%) Asn (N) 71 (4.9%) Asp (D) 125 (8.6%) Cys (C) 2 (0.1%) Gln (Q) 50 (3.5%) Glu (E) 108 (7.5%) Gly (G) 73 (5.0%) His (H) 33 (2.3%) Ile (I) 94 (6.5%) Leu (L) 160 (11.0%) Lys (K) 156 (10.8%) Met (M) 26 (1.8%) Phe (F) 67 (4.6%) Pro (P) 38 (2.6%) Ser (S) 79 (5.5%) Thr (T) 65 (4.5%) Trp (W) 7 (0.5%) Tyr (Y) 59 (4.1%) Val (V) 75 (5.2%)
Amino acid counting Total number: 1448 Positively charged (Arg+Lys): 236 (16.3%) Negatively charged (Asp+Glu): 233 (16.1%) Aromatic (Phe+His+Try+Tyr): 166 (11.5%)	Biochemical parameters Atomic composition: C7507H11924N2038O2239S28 Molecular mass [Da]: 167451.2 Theoretical pI: 8.02 Extinction coefficient at 280 nm [M-1 cm-1]: 126410 / 126535 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.66) good (0.70) acceptable (0.60) good (0.71) excellent (0.84) excellent (0.85)
Alignments (obtained from PredictProtein.org)    There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.

Proof of function

Verification of the suppression efficiency of gRNA
With different target loci have been tested by the usage of a GFP reporter plasmid(pCold-1) with a CSPA promotor. The target sites can be determined by directing the gRNA consisting of 20 bp length against the desired sequence of interest. F2 gRNA with target sites at different distances to the promotor regions proved successfully as potential activation sites (see Table 1 and Figure 3).

Name	Binding Site	Distance to promoter	Sequence	Position
F1	CSPA promoter	15	TGCATCACCCGCCAATGCG	sense sequences
F2	non-coding	68	GCCGCCGCAAGGAATGGTG	sense sequences
R1	CSPA promoter	43	ATTAATCATAAATATGAAA	antisense sequences
R2	non-coding	94	CATCATCCAACTCCGGCAAC	antisense sequences

Table 1: Overview of the tested gRNAs with different binding sites on the GFP pCold-1 plasmid.

Figure 3: Position of the target loci on the GFP pCold-1 plasmid.

To ensure the suppression efficiency of the gRNA, four gRNA sequences targeting different sites of CSPA promoter were designed and transfected into the E. coli strains BL21. Efficient suppression of CSPA promoter in strains BL21 was observed. GFP levels in GFP transgenic strains decrease after inserting a fragment that expresses CSPA promoter gRNA. And the sequence with the best suppression effect was selected for further study.

Figure 4: Results of the GFP-influence under the CSPA promotor only using different gRNAs targeted to CSPA promoter in BL21.. 1-8: transfected with different GFP-gRNA plasmid (CSPA promoter) 9:Control group transfected with GFP plasmid,~27 kDa. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8) at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Silencing capability validation

We next evaluated the effect of tCas9-cibn on suppressing CSPA promoter. GFP expression levels were assayed in strains BL21 after co-transformation with tCas9-cibn and gRNA.

Figure 5: Silencing capability of tCas9-CIBN with gRNA using different gRNAs targeted to CSPA promoter in BL21. 1-8: transfected with different GFP-gRNA plasmid (CSPA promoter) and tCas9-CIBN plasmid(pBAD promoter) 9 10:Control groups transfected with GFP plasmid and tCas9-CIBN plasmid(pBAD promoter), ~27 kDa. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8 10) using 15mM L-arabinose at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Compared with control groups, green fluorescence intensity and mRNA levels were dramatically reduced in groups treated with gRNA and tCas9-cibn. These results suggest that gRNA can specifically guide tCas9 to target upstream of CSPA promoter, thereby to inhibit CSPA promoter to reduce GFP expression levels.

Activation of a fluorescence reporter

Spatially controlled activation of gene expression was achieved in strains co-transfected with the LACE system, a reporter vector containing a gRNA target sequence upstream of CSPA promoter and the eGFP gene. Strains transfected with light-activated CRISPR/Cas9 effector (LACE) and incubated in the dark did not show a significant difference in eGFP levels compared to control groups transfected with empty plasmid. Strains containing the LACE system and gRNA exhibited significantly brighter eGFP fluorescence intensity when illuminated compared to when incubated in the dark.Activation of the eGFP reporter in strains transfected with the gRNA and LACE constructs, the gRNA and tCas9-VP64 expression plasmid or an empty plasmid as a negative control was quantified after 24 hours of illumination or incubation in the dark.

Figure 6: Activation capability of LACE system using gRNA-F2 plasmid and tCas9-CIBN plasmid in BL21. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours using 15mM L-arabinose at 30°C.

Proof of expression

Stationary cultures of BL21 pBAD was subcultured into fresh media and induced for 4 hours or 16 hours using different concentrations L-arabinose. Subsequent purification of protein from the cell-free supernatant and visualization using SDS-PAGE confirms that proteins of the expected size are present in the supernatant and hence most likely successfully secreted by the engineered bacterial strains.

Figure 7: Western blot analysis of tCas9-CIBN protein levels. 1 3 5 7 9 11:Control groups transfected with empty plasmid; 2 4 6 8 10 12: transfected with tCas9-CIBN plasmid (pBAD promoter), ~180 kDa. Stationary cultures of BL21 was subcultured into fresh media and growth for 4 hours or 16 hours using different concentrations L-arabinose. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2758
  Illegal NgoMIV site found at 3667
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 3786
  Illegal SapI.rc site found at 1177
  Illegal SapI.rc site found at 1419
  

[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

[6] Lauren R. Polstein1 and Charles A. Gersbach. (2015). A light-inducible CRISPR/Cas9 system for control of endogenous gene activation. Nat Chem Biol 11(3): 198–200