Difference between revisions of "Team:NTU SINGAPORE/Experiment"

Truncation Project

In vivo application of Cas9 and its variants faces issues with delivery. One of the best method of Cas9 delivery is through recombinant adeno associated virus (rAAV). However, rAAV has a packing limit of approximately 4.7 kB. The Cas9 gene alone is more than 4.1 kB. This leaves very little room for optimal promoter and polyA signal, as well as for sgRNA expression. Recent applications of Cas9 variants fused to effector proteins would also not fit into rAAV vector. Thus, there is strong interest in searching for smaller Cas9 proteins that can perform on par with the well characterized SpCas9.

A continuation of last year’s dCas9 truncation project, this year, we seek to further truncate the Streptococcus pyogenes dCas9 protein to achieve an even smaller protein still capable of sgRNA and target DNA binding. We also mutated key residues to improve dCas9 affinity for DNA, demonstrating partial recovery of our truncated dCas9 (tCas9) towards wild-type activity.

To assess tCas9 ability to bind to sgRNA and target DNA, dCas9 is fused to VPR, a tripartite transcriptional activator. Gene activation activity is then assessed against WT-dCas9-VPR. 2 methods of gene activation are performed. In our proof of concept, exogenous reporter gene activation is performed to rapidly give information of activity of tCas9s. For demonstration, endogenous gene activation is performed, giving a closer approximation of real world application.

Truncation methodology

Prior attempts at truncating tCas9 (Freiburg 2013) were unsuccessful largely due lack of structural information. The publication of the crystal structure of Cas9 (Nishimasu) allowed for identification of domain and subdomains. Based on crystal structure and homology information, Cas9 contains 2 nuclease domains. In dCas9, these domains were mutated to be non-functional.

In this project, we rationally removed domains and subdomains that do not directly contribute to sgRNA binding or target DNA binding. Truncation is largely guided by crystal structure, by selecting domains and subdomains that, when removed, should minimally impact dCas9 folding. A short linker replaces the truncated domain. Truncation is performed at gene level, using Gibson assembly of the dCas9 plasmid. Gibson primers are designed to exclude the truncated region, while introducing the short linker sequence.

Truncations achieved thus far

Of the various truncations tested, truncations in the HNH domain was found to be very well tolerated, likely due to the highly modular nature of the domain. REC2 domain can be truncated, at the cost of around 50% activity – agreeing with literature (Nishimasu). Lastly, a small RuvCIII-2 deletion is also well tolerated.

Although other truncations were also tested, it was found that these 3 truncations combine well together i.e. negative effect on gene activation is minimal when truncations were combined. The triple truncation (∆REC2 ∆RuvCIII-2 ∆HNH, henceforth referred to as ‘∆3ple’) is especially of interest, since it reduces the size of the dCas9 gene to around 3.2kB – on par with the size of Sa-Cas9.

Homology Directed Repair

CRISPR/Cas9 has been widely used as a targeted genome editing tool in human cells. This is due to the simplicity of designing the guide RNA (gRNA), which can be easily designed to target any sequence. This simplicity is especially apparent when compared to previous gene editing tools which rely on protein-DNA interaction, such as meganucleases, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs), which are more difficult to design.

In order to achieve targeted engineering in human cells, Cas9 protein first cuts both strands of the target DNA (guided by the gRNA via complementary base pairing), making a double-strand break (DSB). The DSB is perceived as a negative signal by the cell, and hence is targeted for repair. The cell repairs the DSB by either NHEJ (non-homologous end joining) or HDR (homology-directed repair). In the context of targeted engineering, HDR is preferred since NHEJ can introduce indel mutations, whilst HDR can introduce any donor sequence that we want as long as the donor DNA is flanked by homology regions.

Unfortunately, in living cells, NHEJ occurs more frequently than HDR. Moreover, HDR proteins are expressed only at the S and G2 phases of the cell cycle, since these are the phases when sister chromatids have already formed and can serve as a donor should a DSB occur. This poses a problem if we wish to edit non-dividing cells, such as neurons and myocytes. Therefore, improving the efficiency of the HDR pathway is important to enhance the robustness of CRISPR/Cas9.

In order to improve HDR efficiency, a fusion protein between SpCas9 and Rad52 was developed. Rad52 is one of the HDR proteins involved in mediating ssDNA annealing between homologous sequences and in assisting homologous pairing. The construct was tested in HEK293FT cells, targeting five genes. The construct was transfected concurrently with a GFP donor.

Upon a successful HDR event, the donor will be integrated into the genome and GFP will be produced. Hence, the expression of GFP in the cells indicates a successful HDR event. Next, the cells are analyzed by flow cytometry to determine the frequency of HDR occurrence.

Exogenous Gene Activation

Exogenous gene activation of ZSGreen fluorescent reporter gene is first performed. Quantitative assessment of tCas9 activity is acquired by flow cytometry. 3 plasmids are utilized in exogenous gene activation. The reporter plasmid contains ZSGreen under TREtight minimal promoter preceded by 2X protospacers. The sgRNA plasmid expresses sgRNA under U6 promoter. This plasmid also contains a constitutive mCherry expression, to report successfully transfected cells during flow cytometry. The dCas9-VPR plasmid expresses dCas9-VPR under constitutive CMV promoter. dCas9 can then utilized the sgRNA to bind to protospacer, while the fused VPR greatly upregulates expression of ZSGreen. Cells are transfected and incubated for 48H. Flow cytometry is then perfomed by gating for mcherry positive cells indicated successful transfection. The ZSGreen mean fluorescence is then acquired for these cells, reporting the gene actvation activity of the truncated dCas9-VPR.

EGFR Project

CRISPR-associated (Cas) nucleases from a variety of different bacterial species have been studied for their genome editing capabilities, with each nuclease possessing different characteristics. SpCas9 protein, which is a CRISPR/Cas system found in Streptococcus pyogenes, is one of the most common and widely studied CRISPR-Cas types. Besides SpCas9, other Cas9 proteins as well as the Cpf1 endonuclease are also of interest to us because of their ability to create blunt or staggered double-stranded breaks in target DNA. This makes them potential candidates for gene inactivation, correction or repurpose. In last year's iGEM project, Team NTU performed the efficiency evaluation for 5 Cas9/Cpf1 endonucleases: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Neiserria meningitides Cas9 (NmCas9), Acidaminococcus species (AsCpf1) and Lachnospiraceaebacterium (LbCpf1). In step with the further development and refinement of CRISPR/Cas tools, attempts at introducing the CRISPR/Cas system in vivo though clinical trials have been made. As the application of CRISPR continues to gain traction, we think that there is value in evaluating and determining more suitable CRISPR/Cas systems for therapeutics to facilitate clinical trials for specific diseases.

Therefore, in iGEM 2017, we have decided to investigate the suitability of different CRISPR/Cas systems: SpCas9, SaCas9, AsCpf1 and LbCpf1 endonucleases as therapeutics to target the deletion mutation in the Epidermal Growth Factor Receptor (EGFR) gene related to non-small cell lung cancer. This deletion mutation, namely Del(E746-A750), refers to the in-frame deletion on exon 19 of the EGFR gene. The investigation was performed in the PC9 cell line, which is derived from human adenocarcinoma from lung tissue and harbors the Del(E746-A750) mutation in the EGFR gene, instead of using the Human Embryonic Kidney 293 (HEK293) cell line as in the previous year.

A total of 10 oligonucleotide sequences, which differ by length and sequence, were designed to act as single-guide RNAs (sgRNAs). Four types of CRISPR plasmid constructs (SpCas9, SaCas9, AsCpf1, LbCpf1) were used in the cloning. In order to have a fair comparison amongst all endonucleases, we cloned the sgRNAs onto the same plasmid under the expression of two different promoters, CAG and EF1a.

After cloning, the plasmids were transfected into PC9 cells. Since the plasmids contain the mCherry reporter gene which codes for red fluorescence, successfully transfected cells were sorted via FACS (RFP+) after 24h and 48h. We then extracted the genomic DNA from these cells and PCR amplifed the targeted genomic locus. The amplified products were tested for detection and quantification of indels using T7 endonuclease I (T7E1).

In T7E assay, the PCR products are denatured and reannealed to allow heteroduplex formation between wild-type DNA and CRISPR/Cas9-mutated DNA. T7E1, which recognizes and cleaves mismatched DNA, is used to digest heteroduplexes. The resulting cleaved and full-length PCR products are visualized by agarose gel electrophoresis. The image of the gel is shown under the proof of concept. The diagram below shows the general workflow for T7E assay.