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Revision as of 13:29, 16 October 2018
DNA Base-Editor
Type II CRISPR/Cas System
The bacterial CRISPR/Cas system acts as a form of the adaptive immune system against foreign nucleic acids such as viruses and plasmid. The system is capable of non-self DNA identification, and acquires an excerpt of the non-self DNA – also known as spacers, storing it in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus of the bacterial genome. Subsequently, a CRISPR-associated RNA (crRNA) with the spacer can be expressed and complexed with the CRISPR-associated nuclease (Cas), cleave DNA matching the spacer. Non-self-identification is achieved by an invariant protospacer adjacent motif (PAM), that is found only on the targeted invading DNA, but not integrated into its CRISPR locus.
Various types of the CRISPR/Cas system has been described, with the Type II system being the best studied and used, owing to its relative simplicity. In Type 2 system, the final Cas9 nuclease is a single, multi-functional protein, making it easy to express for genetic engineering purposes. As Cas proteins can only target sequences that has an associated PAM sequence, Streptococcus pyogenes Cas9 (SpCas9) is especially attractive with its relaxed PAM requirement of NGG, compared to variants such as SaCas9 (NNGRRT). Further simplifying the process, it has been shown that the spacer containing crRNA can be fused to an invariant trans-crRNA (tracrRNA), resulting in a single guide RNA (gRNA). Thus, expression of the SpCas9 and a modifiable gRNA is sufficient to target cuts on genomes (Jinek et al., 2012).
One of the first and most well-researched uses of the programmable Cas nuclease in genome engineering. A programmable nuclease can perform cuts on specific loci of the mammalian genome triggering DNA repair machinery that can be taken advantaged of to perform desired modification in the genome. This can potentially allow for the treatment of genetic diseases by correcting aberrant mutations. However, one of the main concerns with Cas9 mediated gene editing is potential off-target cutting. This can lead to indels in unintended loci. Severe aberrances such as large deletions and rearrangements have also recently been described (Kosicki et. Al, 2018). Thus, there is mounting interest in non-permanent solutions for the treatment of diseases such as gene regulation and RNA editing.
Cas9-Fusion Proteins
One of the most exciting applications of Cas9-fusion proteins is in base editing. Fusion of RNA editing APOBEC1 cytidine deaminase, which can also perform DNA editing, allowed for site programmable C to T editing (Komor et al. 2016). Fusion of an evolved TadA Adenine deaminase allowed for A to G editing (Gaudelli et al. 2017). Unlike in HDR directed gene editing, base editors used a nickase cas9 (nCas9) that do not produce a double-strand break (DSB). Thus, it has comparatively higher editing frequency, with much lower undesired mutations such as indels. Base editors may represent a safer method for gene therapy, at least with regards to diseases treatable with single nucleotide changes possible with the current toolset.
APOBEC C-To-T Editing
Design of Experiment
We have previously demonstrated the utility of our tCas9 in the context of VPR fusion for gene activation. Next, we seek to expand on the potential utility of our tCas9 with other fusion proteins. The APOBEC1 fusion as described by Komor et al. (2016) in 3rd generation BE3 stands at close to 700bp, fused to the N-terminus of an nCas9. A 250bp uracil-DNA glycosylase inhibitor (UGI) is also fused to the C-terminus to improve editing frequency. The resulting BE3 construct gene is over 5kB, greatly exceeding the packing limit of rAAV viral vector ideal for in-vivo delivery. Thus, there is value in getting tCas9 variants to work with the BE3 variants.
We tested a model tCas9 variant, ∆Rec3, with the BE3 fusion. The ∆Rec3 fusion was selected for this preliminary experiment for two reasons. First, the ∆Rec3 truncation has been shown to still maintain considerable DNA binding affinity, with over 50% of WT dCas-VPR gene activation in reporter assays (NTU Singapore iGEM, 2017). Second, the Rec3 domain has been shown to play a key proofreading step to allow for HNH cleavage. This is of concern as the high efficiency of both base editors are dependent on HNH nicking of the non-edited DNA strand, promoting repair to match the edited strand. In an experiment, both the tCas9-BE3 fusion protein and its guide RNA are expressed in HEK293t cells, and the target site will be amplified and sequenced to determine the percentage of modification.
Results and Analysis
Results indicate that there is a big drop in base editing efficiency when Rec3 has been truncated (Figure 1). In addition to the poorer DNA binding affinity as observed in gene activation experiments, the loss of Rec3 is likely to have led to the loss of the necessary proofreading step required for HNH nicking, leading to only second generation BE2 functionality for the efficiency of editing.
Figure 1. ∆Rec3-BE3 compared against background (Empty) and WT-BE3.
Conclusion
Results indicated the importance of maintaining HNH nickase activity for optimal base editing efficiency. The removal of Rec3 led to loss of HNH nuclease activity in the context of base editors. In addition, Rec3 participates in interaction with target DNA:gRNA heteroduplex, and the loss of which probably further decreased binding affinity of tCas9. This could explain the drop in base editing efficiency. Moving forward, other truncation sites could be tested to confirm the necessity of function Rec3 and HNH domains.
Results also suggests that base editing is stricter than gene activation in requiring high affinity to the target DNA. This is not unexpected as base editing would likely require longer dwell time for the deamination reaction to be catalysed. The improvements achieved with our ∆3ple in this year’s project could potentially solve this issue, although HNH may have to be replaced to maintain nickase function.
Experimental Design
The adenine base editor (ABE) has been evolved from a transfer RNA adenosine deaminase (TadA) that capable of converting A•T to G•C base pairs to treat diseases caused by single-base mutations in the genome such as sickle-cell anemia. The ABE can then be fused to a dCas9 to target a desired site to edit the DNA guiding by sgRNA.
However, overexpression of a protein can lead to toxicity issue, especially with the large size of dCas9. In order to have a tight control of gene regulation, we construct two plasmids where TadA*-dCas9 is expressed by arabinose under pBAD promoter and sgRNA is expressed by IPTG under lac operator. The TadA*-dCas9 plasmid consists of chloramphenicol resistance while the sgRNA plasmid consists of a gentamycin resistance and a kanamycin resistance with a premature stop codon.
Figure 2. Two constructs to be transformed into E. coli
In order for the bacteria to survive on Chloramphenicol + Gentamycin + Kanamycin condition, the TadA*-dCas9 need to restore the premature stop codon of Kanamycin.
We transformed bacteria BW25141 with these two plasmids and used 2 hours of LB recovery under four conditions: no induction, 100mM arabinose, 500μM IPTG, and 100mM arabinose + 500μM IPTG. The samples were then spread on a Chloramphenicol + Gentamycin plate for positive control and a Chloramphenicol + Gentamycin + Kanamycin plate for selection.
Results and Analysis
The result of the transformation are shown in figure below. Colonies grown on chloramphenicol+gentamycin plate show that the host cells contained TadA*-dCas9 and sgRNA plasmids, but colonies only grown on chloramphenicol + gentamycin + kanamycin plate prove the tightness of regulation of both promoters.
Figure 3. Survival of colonies after transformation and induction
As seen from above, similar growth was observed on control plates without the selection pressure of Kanamycin, suggesting a similar transformation efficiency. However, in the presence of Kanamycin, only the experiement with both inducers added showed siginificant growth, suggesting that both constructs are necessary for bacteria to grow in Kanamycin and proving the editing activities of the TadA*-dCas9 construct. Such a growth can be more rigorously quantified as survival frequency, defined as the ratio of the number of colonies on the Chloramphenicol + Gentamycin + Kanamycin plate to that on the Chloramphenicol + Gentamycin plate. We also collaborated with NUSGEM this year to validate our TadA*-dCas9 construct.
Conclusion
From both experiments, we can conclude that the fusion protein between base editors and Cas9 indeed functioned as expected to perform either single base change or base pair change. However, the editing efficiency of these proteins remains to be further explored and optimized. It is also worth noting here that different approaches can be employed to quantify base editing activities. In the tCas9-BE3 experiment, direct sequencing was used to determine the percentage of edited DNA while in the TadA*-dCas9 experiment, a more intuitive reporting system of measuring colony formation was employed. This shows our creativity in designing our experiment, which we should continue to innovate and develop.
References
1. Chen, J. S., Dagdas, Y. S., Kleinstiver, B. P., Welch, M. M., Sousa, A. A., Harrington, L. B., … Doudna, J. A. (2017). Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature, 550(7676), 407–410.
2. Komor, A., Kim, Y., Packer, M., Zuris, J., & Liu, D. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420-424. doi: 10.1038/nature17946
3. Gaudelli, N., Komor, A., Rees, H., Packer, M., Badran, A., Bryson, D., & Liu, D. (2017). Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 551(7681), 464-471. doi: 10.1038/nature24644