View source for Template:CasX

===CasX: Usage and Biology===

This part contains the construct used to express the DpbCas12e CRISPR nuclease under the CasX family. 

CasX is a subfamily of CRISPR endonucleases that refers to two enzymes–DpbCas12e and PlmCas12e–derived from homologous systems, Deltaproteobacteria and Planctomyces. While PlmCas12e shows higher gene editing efficiency in vivo, Dpbcas12e shows much higher cleavage activity in vitro. CasX is a relatively small CRISPR protein with a size of <1000 amino acids. It has a dual RNA guide/activator composed of a 60-70nt tracrRNA and a crRNA. Unlike Cas9 proteins it does not have a blunt cutsite; as a member of the family of Cas12 nucleases, it has a sticky-ended cutsite formed at 18nt and 22nt relative to the spacer region (although this has been contested among different papers). In addition, CasX has relatively low trans-cleavage activities of ssDNA strands as characterized in Liu et al, 2019.

===Characterization and Verification===

The complete CasX protein construct is 156.25 kDA in size. 

We transformed two constructs expressing CasX, one we designed on our own titled CasX_DualHis_Thrombin referred to as CasX0) and pJJGL001 from AddGene (referred to as CasX1). Our variant is Biobrick compatible as it does not contain EcoRI cutsites, and the dual His tag was added to assist in purification methods. Plasmid maps for both constructs transformed are shown below: 

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<img src="https://static.igem.wiki/teams/5099/parts-collection-figures/basic-reporter-gates/casx1-plasmidmap.png" 
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Constructs were transformed into BL21(DE3) E. Coli comp cells from NEB. 5 colonies grew for CasX0, while 6 colonies grew for CasX1. We tested varying induction conditions for the protein. Colonies were picked and induced at the following conditions. Induction was carried out at an OD of 0.4-0.6 and duration was overnight (12-16 hours) at 1mM of IPTG. We tested induction at 37ºC and 25ºC for all the colonies with the following results: 

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Since the band was not more pronounced in the 37ºC growth condition and the protein was large, we decided to induce overnight at RT (25ºC) continuing forward. 

Furthermore, we tested different sonication protocols for the protein using varying sonication (more can be seen on our Results/Experimental page). We found that sonicating for 10 seconds on/off continuously for 5 minutes yielded the best results in induction (condition 3).  

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Afterwards, we moved to His-tag purification of the protein with NiNTA columns, and found the protein purified best at 25ºC. Elutions after His-tag purification are shown below, with our successfully expressing colony being CasX1.5. This also helped to verify the presence of His-tagged protein in the purified sample, which we validated by Western blot. 

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[Insert Western image here] 

Optimal expression conditions were determined to be 22ºC, 16 hours, 1mM IPTG in LB. After extraction and purification, proteins were flash-frozen in liquid nitrogen for further use in cleavage and fluorescence assays.