Part:BBa_K1774000
CRISPR-Cas9 contaiment device repressed by arabinose and tryptophan
Overview
This is a prototype containment device which aims to control accidental leakage of GMM once it escape from medium containing arabinose and tryptophan. The device consists of two modules, an arabinose repressible cas9 expression module and a tryptophan repressible gRNA expression module. Since we plan to experiment this device on E. coli BL21 (DE3), we have designed a gRNA that target the chassis only. Under the presence of sufficient amount of arabinose and tryptophan the device remain silent, however when bacteria escape to environment without arabinose and tryptophan, Cas9 and gRNA targeting DNA polymerase III alpha subunit (dnaE) will be expressed. Subsequent cleavage of the targeted sequence may hinder replication of bacteria.
CRISPR-Cas9
Our device utilize CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated), an bacterial adaptive immune system originated from Streptococcus pyogenes SF370 which might play an important role in combating bacteriophages by destroying phage DNA. The wild type system constitute of three essential components. (1) Cas9 endonuclease with two DNA cleavage sites (RuvC and HNH). (2) A non-coding CRISPR array composes of series of repeat-spacer which contain sequence derived from invading DNA. Transcription and processing of the CRISPR array produce number of crRNA carrying 20nt complementary to the target sequence. (3) A trancRNA (trans-activating crRNA) which form a RNA duplex (trancRNA:crRNA) with crRNA that direct binding of Cas9 to target DNA. Base pairing of tracRNA:crRNA with target sequence immediate upstream of protospacer adjacent motif (PAM), in this case 5'-NGG-3' initiates double strand break by RuvC and NHN nuclease domain in Cas9 endonuclease.
In order to simplify the device, we have reprogram the system with single guide RNA (sgRNA) instead of using the CRISPR array and tracRNA. This reprogramming is also widely adopted in many genome editing applications. Moreover, this construct also make use of codon optimized Cas9 (BBa_K1774001) for better translation efficiency in E. coli.
How the device work
OFF state -- With arabinose and tryptophan
1. Arabinose activate pBAD by repressing AraC
2. Tryptophan repress pTrp by activating TrpR
3. Activation of pBAD initiate expression of CI which repress expression of Cas9
4. Repression of pTrp lead to low expression of gRNA
5. The expression of CRISPR-Cas9 system is repressed by the presence of arabinose and tryptophan
ON state -- Without arabinose and tryptophan
1. Net consumption of arabinose and tryptophan repress pBAD and activate pTrp
2a. Repression and degradation of CI activate Cas9 expression
2b. Activation of pTrp initiate transcription of gRNA
3. Expression of both Cas9 and gRNA lead to cleavage of dnaE, hence inhibit replication and perhaps lead to cell death
References
[1] Chylinski, K., Le Rhun, A., & Charpentier, E. 2013. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol. 10:726–737
[2] Ran, F.A. et al. 2013. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8:2281–2308.
[3] Sander, J.D. & Joung, J.K. 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32:347–355.
[4] M. Jineket al. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337:816.
[5] Deltcheva, E. et al. 2011. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 471:602–607.
[6] Bortesi, L. & Fischer, R. 2015. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances. 33:41–52.
[7] Schleif, R. 2003. AraC protein: a love-hate relationship. BioEssays. 25:274-282.
[8] Gunsalus, R.P. and Yanofsky, C. 1980. Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor. proc. Natl. Acad. Sci. USA. 77;12:7117-7121.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 7081
Illegal SpeI site found at 7089 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1247
Illegal NheI site found at 6532
Illegal NheI site found at 6555
Illegal SpeI site found at 7081
Illegal SpeI site found at 7089 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1187
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 7081
Illegal SpeI site found at 7089 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 7081
Illegal SpeI site found at 7089
Illegal AgeI site found at 979 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Illegal SapI site found at 5785
Illegal SapI.rc site found at 6761
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