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===CasX===
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===CasX: Usage and Biology===
  
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The Nucle.io project aims to provide rapid, point-of-care diagnostics that undercut current wait times for the return and analysis of laboratory test results such as blood culture (3 days) and PCR (1 day), by performing both diagnostic amplification and result computation in one reaction. This allows clinical decision making to happen on a faster timescale in emergency medical settings where time is of the essence. Sepsis is a disease responsible for 20% of global deaths. Each hour of delayed treatment leads to an 8% increase in mortality. When infection is treated early with accurate antibiotics, downstream complications such as organ failure can be prevented. The Nucle.io diagnostic uses a modular approach to achieve both accuracy and speed, leveraging the CasX protein to perform mRNA-based detection or a strand displacement cascade to perform mRNA amplification. The downstream module (computation) applies the Winner-Take-All neural network (WTA NN), which is a DNA computing architecture using toehold-mediated strand displacement reactions to analyze profiles of nucleic acids developed in Neural network computation with DNA strand displacement cascades (Qian et. al, 2011).
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This part contains the construct used to express the DpbCas12e CRISPR nuclease under the CasX family.  
  
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===Usage and Biology===
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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.
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CasX is a broad name for a family of Cas proteins later reclassified under Cas12e. McGill iGEM uses DpbCas12e isolated from Deltaproteobacteria due to high in-vitro cleavage activity (variant PlmCas12e displays low activity in-vitro). CasX uses a dual-RNA guide effector composed of a tracrRNA and crRNA. The tracrRNA and crRNA guide complex contains a scaffold stem containing a bubble, an extended stem, and a triplex region.  
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===Characterization and Verification===
 
===Characterization and Verification===
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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:  
 
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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Afterwards, we moved to His-tag purification of the protein with NiNTA columns, and found the protein purified best at 25ºC. 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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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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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.  
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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.
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Latest revision as of 12:34, 23 September 2024

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:


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:


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).

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.

[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.