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Revision as of 07:57, 1 November 2017
CRISPR Cas9
PACE for the Evolution of Endonucleases
Introduction
Many of nowadays most threatening diseases are caused by mutations, epi mutations or other changes in the genome. Although medical research was always supplied by innovations in biological research and especially by the field of genetics, which developed rapidly during the last decades, there are still many diseases that cannot be cured or even treated adequately. Recently, the CRISPR/Cas9 technology raised hope of the scientific community to treat genetic disorders. This technique has dramatically simplified the way genomes can be manipulated. However, there are still many challenges to be surpassed. Cas9 and related endonucleases are enzymes, which are able to induce double strand breaks in the genome. Importantly, they only cut specific sequences to which they are guided by a so called guideRNA (gRNA). A gRNA consists of a 3' scaffold, which is obligatory for the binding of the Cas9 enzyme, a protospacer sequence, and 20 nucleotides at the 5'-end that are complementary to the target DNA. Once the Cas9 endonuclease binds to the DNA, it cleaves three nucleotides upstream of the protospacer 3'-end. This system allows to target virtually any position in any genome. However, there is one major restriction in the applicability of this system. Only sequences can be targeted that carry a specific recognition motif directly downstream of the spacer, the protospacer adjacent motif (PAM). In case of Cas9, the consensus PAM is NGG![](
https://static.igem.org/mediawiki/2017/f/f4/T--Heidelberg--Team_Heidelberg_2017_pampacEABSTRACT1.jpeg)
Figue 1: Principle of PACE for the Evolution of Cas9
The production of phages is linked to the fitness of Cas9 via the phusion to rpoZ When the gRNA targets the fusion protein to a sequence upstream of the promoter, the rpoZ is able to recruit the transcription machinery and therefore activates genVI expression and subsequently phage production. When the PAM upstream of the spacer is altered, Cas9 cannot bind anymore and the expression stops. In context of PACE the nuclease mutates. Mutants of Cas9 with that can bind to the new PAM, activate ProteinVI production. This provides a fitness benefit. With the generation of randomized PAM libraries, the overall PAM specificity can be decreased, which is of great interest for many gene editing applications
The Idea
To prove our hypothesis, we planned a circuit for the directed evolution of PAM specificity of Cas9. The main challenge was to link transcription activation to the binding of Cas9. We chose a system, which contains a dCas9 fused to a RNA polymerase Ω subunit (rpoZ)Phage Based in vivo Eolution with GeneVI
One of the major challenges in the context of transcription activation with help of the rpoZ is leaky expression. This is a setious problem for PACE, because if geneIII is expressed prior to phage infection leads to infection the bactirial cell turns resistant![](
https://static.igem.org/mediawiki/2017/6/6b/T--Heidelberg--Team_Heidelberg_2017_graphical_abstract_JMu.png)
Design of the Accessory Plasmids for the Evolution of Cas9
The AP consists of five subparts that are devided by homology regions for Gibson assembly (numbers). It carries an expression cassette for the transcription of a gRNA (between 1 and 5). GeneVI (2-3) is under control of a that can be activated by the Cas9-rpoZ in context with the respective gRNA. luxAB accounts as a reporter for fluorescent readout of geneIII activation (3-4). The whole plasmid can be produced with different origins of replication (4-5) to modulate the copy number and by exchanging the geneVI part with the RBS.
Our Accessory Plasmids for PACE of Endonucleases The different accessory plasmids that were cloned in the context of this project are shown. The constructs differ in their copy number and the strength of their RBSs.
Puri-ID | AP | Regulatory Sequence | RBS of geneIV | Origin of replication | gRNA cassette | PAM |
---|---|---|---|---|---|---|
821 | AP_Cas9_pSC101_NNNN_SD8_GVI | minimal promoter downstram of the dCas9 target sequence | SD8 | pSC101 | gRNA expression cassette | NNNN |
822 | AP_Cas9_pSC101_NNNN_sd8_GVI | minimal promoter downstram of the dCas9 target sequence | sd8 | pSC101 | gRNA expression cassette | NNNN |
823 | AP_Cas9_pSC101_NNNN_sd6_GVI | minimal promoter downstram of the dCas9 target sequence | sd6 | pSC101 | gRNA expression cassette | NNNN |
824 | AP_Cas9_pSC101_NNNN_SD4_GVI | minimal promoter downstram of the dCas9 target sequence | sd4 | pSC101 | gRNA expression cassette | NNNN |
825 | AP_Cas9_pSC101_NNNN_sd2_GVI | minimal promoter downstram of the dCas9 target sequence | sd2 | pSC101 | gRNA expression cassette | NNNN |
826 | AP_Cas9_pSC101_NGAN_SD8_GVI | minimal promoter downstram of the dCas9 target sequence | SD8 | pSC101 | gRNA expression cassette | NGAN |
829 | AP_Cas9_pSC101_NGAN_SD4_GVI | minimal promoter downstram of the dCas9 target sequence | sd4 | pSC101 | gRNA expression cassette | NGAN |
830 | AP_Cas9_pSC101_NGAN_sd2_GVI | minimal promoter downstram of the dCas9 target sequence | sd2 | pSC101 | gRNA expression cassette | NGAN |