Our main goal was to create a novel CRISPR-based system that can detect disease causing mutations without the use of DNA sequencing. We focused our attention on two different approaches:
1) CRISPR-Cas9 – RFLP: The idea is to use the wild type CRISPR-Cas9 system with appropriate gRNAs. These complexes can cut in vitro PCR products only if a given mutation is present. The output can be visualized on a simple agarose gel without the need of sequencing.
2) Paired CRISPR-dCas9 sensors: Split fragments from a reporter protein can be fused to a pair of dCas9 enzymes. If both inactivated nucleases recognize their specific targets, the split protein fragments will be in close enough proximity to each other, re-assembling the reporter molecule. In this case, our system will generate a specific output signal – fluorescence, luminescence or a colorful enzymatic reaction.
In order for it to be time and cost efficient, we focused our efforts on approach one. The design of paired dCas9 sensors will, hopefully, be completed next year due to severe financial limitations.
STEP-BY-STEP EXPLANATION OF OUR DESIGN
Selection of model disease: We selected cystic fibrosis as a model disease for our system, since it is the most common genetic condition in Bulgaria and Europe as a whole. Many Bulgarian patients that live in remote regions and/or small towns and villages are diagnosed far later, compared to other European countries. In the light of these facts, a novel system for molecular diagnostics of cystic fibrosis will prove incredibly useful. Moreover, a single mutation (del F508) in the CFTR gene is responsible for approximately 70% of the cases. This variant affects three nucleotides located right next to a NGG region that can serve as a PAM site for different CRISPR-Cas9 systems. This makes the del F508 mutation the best candidate for our system.
Design of gRNAs: The specific location of the delta F508 mutation allowed us to design two different gRNAs: one that targets the wild type locus and another to recognize the mutated DNA sequence. Both molecules differ in 3 nucleotides located just next to the PAM region. According to literature, differences in this region have severe effect on the ability of Cas9 to recognize and cut its targets.
Design of gRNA expression vectors: To test our system in vivo, we cloned both gRNAs into a pSB1K3-gRNA vector (a kanamycin-resistant version of part BBa_K2515002). Since the combination of a gRNA targeting a sequence in the E. coli genome and Cas9 is lethal for these bacteria, we checked both our constructs using a BLAST search against the E. coli genomic sequence. The gRNA that targets the delta F508 mutation showed no significant similarities, so we concentrated on it for our in vivo experiments. For in vitro studies, the off-target effects are not a concern.
Cas9 system: We used a plasmid for Cas9 expression from Addgene. It has a weak constitutive promoter and a chloramphenicol resistance cassette. Our gRNA construct was then transferred to the pCas9 strain via the process of transformation, followed by selection on both antibiotics.
Cas9 chromosome integration system: In order to reduce the total plasmid number, we created an E. coli strain with a Cas9 expression cassette integrated into its genome. The Cas9 expression is under the control of an arabinose-induced promoter. The Tn7 transposon-based system was selected specifically for the integration step.
In vivo check of the gRNA specificity: The specificity of the gRNA is critical for the proper functioning of our del F508 detection system. To experimentally validate this aspect, we used a gBlock fragment with the corresponding CFTR gene region, containing the mutation of interest. This part was cloned into our vector, pSB1K3-gRNA, and the resulting construct was validated via colony PCR. Next, we transferred that plasmid into pCas9 containing competent cells. After a two-hour period of outgrowth at 37 o C, aliquots were plated on petri dishes with chloramphenicol and chloramphenicol plus kanamycin. The colony numbers were analyzed on the next day. If the gRNA is active against its target sequence, one would expect no (or just a few) colonies on the dish with kanamycin, since Cas9 will eliminate the gRNA-producing vector. If the gRNA cannot recognize the analysed target, many colonies will grow on the plate, since the transformation step is performed with a purified super-coiled plasmid. Using this approach, we aimed to design a gRNA molecule that targets the mutated version of the CTFR gene sequence at one hand, and cannot recognize the wild type on the other.
In vitro mutation detection: Specific PCR primers were designed to be able to amplify a part of the CTFR gene (approximately 1 kb in size). An in vitro Cas9 digestion reaction is performed, using purified Cas9 nuclease protein and a specific gRNA. The results of this reaction are analyzed on agarose gel electrophoresis.
EXPERIMENTAL PLAN TO TEST OUR DESIGN
Design of gRNAs: Two specific 20-base long gRNAs were manually designed using the closest possible PAM region, located just next to del F508. They will be analyzed with in vivo and in vitro experiments.
Cloning of the gRNAs: We used the vector BBa_K2515002, designed by iGEM Bulgaria 2017 team. The gRNA expression cassette was digested with EcoRI and PstI and then subcloned into a pSB1K3 standard vector to obtain the pSB1K3-gRNA expression system. Positive clones were verified by colony PCR.
gRNA activity and specificity: These properties were analyzed via the addition of the targeting region (with and without the mutation) to the gRNA expression vectors. If the gRNA is active against the given sequence, the plasmid is eliminated when the bacteria have pCas9 inside them.
In vitro mutation detection: PCR amplified regions from the CTFR gene from patients with del F508 and healthy controls will be treated with Cas9 + gRNA in vitro. The result can be monitored on agarose gel.