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Project Inspiration

In recent years, there has been much interest surrounding human DNA editing. However, major issues regarding human DNA editing remain. This includes safety concerns from possible off-target effects as well as ethical and regulatory considerations.

DNA editing is a permanent way of correcting disease-causing single-nucleotide polymorphisms (SNPs). However, side effects from potential off-target activity would be permanent and heritable (if the germline was affected). In contrast, RNA editing allows for correction of disease-causing SNPs and is not permanent or heritable. Feedback from members of the public, academics and doctors collected in the early phase of our project design helped us realize that there is a need for a single-nucleotide editing platform that is safe, specific and efficient. Hence for iGEM 2019, we sought to further improve on our dCasRx-ADAR2_DD fusion protein from last year to obtain a fusion protein that has higher RNA editing activity and higher specificity (low off-targets). We also aim to investigate the effects of dCasRx-ADAR2_DD-mediated RNA editing on open and structured RNA and examine the multiplex editing of our dCasRx-ADAR2_DD construct. Our interviews with doctors and academics also led us to incorporate a new characterization parameter to address safety concerns of RNA editing - off-target editing of our constructs.

Project Description

RNA Editing with dCasRx-ADAR

Cas13 enzymes

The CRISPR/Cas system is a prokaryotic adaptive immune system that provides protection against foreign nucleic acids. DNA-targeting CRISPR enzymes such as Cas9 and Cas12a have emerged as powerful tools to edit DNA sequences and modify gene function. However, its promise also raises safety and ethical concerns associated with human DNA editing.

In recent years, bioinformatic screens have uncovered new Cas enzymes that target RNA called Cas13. There are currently 4 known members in the Cas13 family: Cas13a, Cas13b, Cas13c and Cas13d. Cas13 enzymes are smaller than Cas9 enzymes at approximately 930 amino acids long, making them ideal candidates for in vivo AAV delivery. All Cas13 members contain 2 higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains, which is responsible for RNA cleavage. Cas13 enzymes have a preference for targets with protospacer flanking sites (PFSs), but do not require them for biotechnological use in mammalian cells. Compared to DNA editing with Cas9 enzymes, targeted RNA editing with Cas13 enzymes is a safer alternative as edited RNA is easily degraded and not heritable, making any potentially deleterious edits temporary and reversible.

ADAR enzymes

3 adenosine deaminase acting on RNAs enzymes (ADARs) exists in humans (ADAR1 to 3). However, only ADAR1 and ADAR2 have been shown to perform RNA editing. ADAR3 lacks deamination activity and is less important in RNA editing. ADAR1 and 2 are both expressed in most tissues whereas ADAR3 is found exclusively in the brain.^[1] ADAR1 mainly edits repetitive sequences, while ADAR2 edits non-repetitive coding sequences.^[2-4] Hence, ADAR2 is of greater relevance to our work in correcting missense mutations.

Both ADAR1 and ADAR2 contain an N-terminal double-stranded RNA-binding domain and a C-terminal deaminase domain.^[2] The ability to edit RNA resides in the deaminase domain of the ADAR enzymes, which catalyses the hydrolytic deamination of adenosine (A) to inosine (I) in doubled-stranded RNA (dsRNA). During translation, I is read as a guanosine (G), which functionally results in an A>G conversion. This single nucleotide change can result in a codon that codes for a different amino acid, thereby changing the protein product that is translated. For example, disease-associated proteins from a G>A mutation can be corrected back to wild-type through A>I(G) editing.

Mechanism of Cas13-ADAR RNA Editing

2 members of the Cas13 family are of particular interest to us: Cas13b and Cas13d. In one study, Cox et al. (2017) fused the catalytically inactive Cas13b ortholog from Prevotella sp. P5-125 (dPspCas13b) to human ADAR2 deaminase domain containing a hyperactivating mutation E488Q (ADAR2_DD).^[3] ADAR enzymes selectively edit RNA containing their preferred sequence motifs. The E488Q mutation replaces glutamic acid at position 488 with glutamine and relaxes the sequence specificity of ADAR2. This dPspCas13b-ADAR2_DD system is referred to as RNA Editing for Programmable A to I Replacement (REPAIR), and demonstrates efficient and robust targeted A>I(G) RNA editing in mammalian cells. Next, they mutated T375G in REPAIRv1, and termed it REPAIRv2. In another study, Konermann et al. (2018) showed that catalytically inactive Cas13d derived from Ruminococcus flavefaciens XPD3002 (nicknamed CasRx) can to be targeted to specific RNA elements to modulate alternative splicing events in vivo with high specificity.^[1] Hence, CasRx could be engineered for programmable RNA targeting by fusing it to ADAR2_DD.

Here, our constructs consist of a catalytically inactive CasRx (dCasRx) fused to ADAR2_DD. For iGEM 2019, we have chosen to improve upon last year's dCasRx-ADAR2_DD fusion protein by inserting ADAR2_DD into internal sites within dCasRx and mutating key residues in ADAR2_DD to improve RNA editing efficiency and specificity. A gRNA (ssRNA) is designed with complementary sequence to the mRNA target (ssRNA). Upon binding of gRNA to the mRNA target, dCasRx-ADAR2_DD is recruited to the target and a dsRNA substrate is formed for ADAR activity. To specify the A to be edited, a cytosine (C) mismatch is designed on the gRNA, which causes a bulge to be formed. The bulging A is then preferentially targeted by ADAR and converted to I. Previous work by our advisors at the Genome Institute of Singapore (A*STAR) has demonstrated that decreasing the gRNA spacer length to 26 base pairs allows for a higher percentage of RNA editing. Hence, all gRNAs designed for our experiments are 26 base pairs in length and contain an internal cytosine mismatch to the adenosine on the target site to specify the adenosine for deamination by ADAR2_DD. dCasRx fused to ADAR2_DD containing the E488Q mutation (CasRx v1, also BBa_K2818001 by Team NTU-Singapore 2018) and dCasRx fused to ADAR2_DD containing the E488Q and T375G mutation (CasRx v2) were used as a reference to compare the editing activity of our dCasRx-ADAR2_DD constructs to.

Experimental methods

Based on Cox et al. (2017), we have established 2 methods to measure and characterize the RNA editing activity of our constructs. First, we developed a luciferase reporter assay to assess exogenous RNA editing activity. Next, we performed Sanger sequencing and Amplicon sequencing (Illumina) of endogenous target mRNA to assess endogenous RNA editing activity.

In our luciferase reporter assay, a plasmid (*Rluc) encodes a Renilla luciferase gene in which a guanosine (G) is replaced by an adenosine (A). This disrupts the luciferase gene, causing it to be nonfunctional (W60X mutation; tryptophan to a stop codon at position 60). When the *Rluc plasmid is co-transfected along with our dCasRx-ADAR2_DD constructs and a gRNA targeting *Rluc, ADAR2_DD converts A>I(G), which restores the luciferase gene and generates luminescence (X60W; stop codon to tryptophan). This allows for RNA editing activity to be quantified.

For Sanger and Amplicon sequencing, HEK293FT cells were co-transfected with plasmids encoding dCasRx-ADAR2_DD and targeting gRNA. HEK293FT ADAR1 knockout cells were co-transfected with plasmids encoding dCasRx-ADAR2_DD and non-targeting gRNA. Following a 48-hour incubation period, total RNA of the cells were extracted and converted to cDNA. Next, amplification of on-target editing and off-target editing sites were performed with primers targeting these sites to determine RNA editing efficiency and specificity of our dCasRx-ADAR2_DD constructs. PCR products were then processed for Sanger and Amplicon sequencing. To quantify editing of on and off targets for Sanger and Amplicon sequencing, we used the formula of % editing = PeakG/(PeakG + PeakA).

Analysis of amplicon sequencing data

References

1. Konermann S, Lotfy P, Brideau N, Oki J, Shokhirev M, Hsu P. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell. 2018;173(3):665-676.e14.
2. Wang Y, Beal P. Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method. Nucleic Acids Research. 2016;44(20):9872-9880.
3. Cox D, Gootenberg J, Abudayyeh O, Franklin B, Kellner M, Joung J et al. RNA editing with CRISPR-Cas13. Science. 2017;358(6366):1019-1027.
4. Tan M, Li Q, Shanmugam R, Piskol R, Kohler J, Young A et al. Dynamic landscape and regulation of RNA editing in mammals. Nature. 2017;550(7675):249-254.