Team:Heidelberg/CRISPR

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   CRISPR Cas9|
   PACE for the Evolution of Endonucleases|

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       CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) related endonucleases revolutionized the field of genetic engineering as they simplified a sequence specific DNA targeting based on Watson Crick base pairing. They are currently used for various applications, for instance genome editing JM_1, transcription regulation JM_2 or RNA editing JM_3.The development of endonucleases with altered functions would be a benefit for the whole field of genome engineering. For example, the availability of a multiplicity of PAM sequences would allow to edit any sequence regardless of the target sequence itself.
Besides, rational design approaches such as directed evolution are promising approaches for the development of innovative CRISPR/Cas tools that can push forward the gene editing applicability.
With this subproject, we want to demonstrate that the PAM-sequence of endonucleases can be evolved with the PACE method. For our evolution circuit, we linked the transcription of geneIII to the fitness of a catalytically inactive Cas9 - dCas9. In particular, we planned to alter the PAM specificity of dCas9. Therefore, we used a fusion of the dCas9 and the RNA polymerase Ω subunit (rpoZ), which renders transcription activation in E. coli possible.

    
    




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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 Jinek2012. The past few years, much effort has been put into the development of the CRISPR/Cas9 technique. In order to create even more sophisticated systems, many attempts have been made to modify the CRISPR-associated (Cas) endonucleases, for example the development of a catalytically inactive dCas9 RN141 or nickases Mali2013x.
Attempts to change endonuclease activity by directed evolution have already been made 
JM_5Gao2017. We wanted to show that *in vivo* directed evolution of endonucleases is possible as well. This approach would overcome the limitations of the evolution based on rationalities and would offer new tools for the genome engineering research area.




  

    
    

      
                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 genIII 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 ProteinIII 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.
We chose a system for the transcription activation, which contains a dCas9 fused to a RNA polymerase Ω subunit (rpoZ). In our scenario, the nuclease targets a region upstream of a minimal promoter. In case the dCas9 is able to bind the DNA, the fused rpoZ recruits the transcription machinery and geneIII is expressed. By changing the PAM sequence or generating PAM libraries, it is possible to induce a selection pressure on the randomly mutating protein. As a result, proteins with a weaker PAM specificity evolve, which can be used, no matter if the NGG motif is present exactly at the desired position.
Of course, this first circuit was designed according to our cloning standard by Gibson assembly.

  
Design of the Accessory Plasmids


All parts, which were necessary for the assembly of Accessory Plasmids were generated by PCR with the respective homology regions in the extensions. Subsequently, they were assembled by CPEC. All APs carry a bicistronic operon for the expression of geneIII and luxAB as luminescent reporter downstream of the promoter, described above. An expression cassette with the required gRNA under the control of a constitutive promoter is located on the same plasmid. APs varying in the copy number of their origins of replication and the strength of the RBS upstream of geneIII were cloned %%tabref:<1>; %%tab:1; Plasmids, that were cloned for the evolution of PAM specificity, the plasmid names, and the functional parts they consist of are shown.

  


  

    
    

      
                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).  GeneIII (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 geneIII part with the RBS.

References