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.
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.
Homology Directed Repair
CRISPR-Cas9 has been used widely as a targeted genome editing tool in human cells. This is owing to its simplicity in designing the guide RNA (gRNA), which can be designed easily to target any sequence. Especially if it is compared to the previous tools that 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 a targeted engineering in human cells, Cas9 protein first cut both strands of 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 need to be repaired. The cell then will repair the DSB by either NHEJ (non-homologous end joining) or HDR (homology-directed repair). In the context of targeted engineering, HDR is more preferred since NHEJ can introduce indel mutations, while HDR can introduce the 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 S and G2 phase of cell cycle, since naturally these are the phases when sister chromatids already synthesized and can serve as a donor in case a DSB happens. These facts are problems if we are to edit cells that will not undergo cell cycle anymore, such as neurons and myocytes. Therefore, improving the efficiency of HDR pathway is important so that the CRISPR-Cas9 is more robust.
Methodology
In order to improve the HDR efficiency, a fusion protein between SpCas9 and Rad52 was developed. Rad52 is one of the HDR proteins which is involved in mediating ssDNA annealing between homologous sequence and assisting homologous pairing. The construct was tested in HEK293FT cells, targeting five genes. The construct was transfected concurrently with a GFP donor.
Upon the successful HDR event, the donor will be integrated into the genome and GFP will be produced. Hence, a successful HDR is indicated by the expression of GFP in the cells. Then, the cells will be analyzed by flow cytometry to determine the frequency of HDR happening.
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. For iGEM, constructs with SpCas9, SaCas9, AsCpf1 and LbCpf1 systems, derived from Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Acidaminococcus species and Lachnospiraceaebacterium respectively, will be investigated. 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 propose that there is value in evaluating and determining more suitable CRISPR systems for therapeutics to facilitate clinical trials for specific diseases.
In this project, we have decided to investigate the suitability of different CRISPR proteins (SpCas9, SaCas9, AsCpf1 and LbCpf1) as therapeutics to target two significant mutations in the Epidermal Growth Factor Receptor (EGFR) gene related to non-small lung cancer, namely the Del(E746-A750) and L858R mutation. Del(E746-A750) refers to the in-frame deletion on exon 19, while L858R refers to the missense point mutation in codon 858 of exon 21, resulting in substitution of lysine with arginine.
Methodology
A total of 30 oligonucleotide sequences served as guide RNA (gRNA) were designed to act as sgRNAs. Four types of CRISPR plasmid constructs (SpCas9, SaCas9, AsCpf1, LbCpf1) were used in the cloning. In order to have a fair comparison among all endonucleases, we cloned them onto the same plasmid under the expression of two different promoters, CAG and EF1a.
After cloning, transfection of plasmids into specific cell lines were conducted, in which plasmids that target L858R mutation were transfected into H3255 cells harbouring the L858R mutation, and plasmids that target Del(E746-A750) were transfected into PC9 cells harbouring the deletion mutation. We also transfected both types of plasmids into A549 cells with the wild-type EGFR gene to serve as negative controls. Since the plasmids contain mCherry reporter gene, successfully transfected cells after 24h and 48h were sorted via FACS (RFP+). We then extracted the genomic DNAs from these cells and PCR amplify 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.
