Team:WPI Worcester/Description
Project Description
Typical gene editing strategies using CRISPR/Cas9 rely upon homologous recombination that is inefficient and often results in indel mutations. Recently, a technique was described to induce direct C->U editing in DNA targets; however the utility of this new tool is limited to a single mutation and still relies on endogenous DNA repair. We propose to use CRISPR-based RNA targeting and dCAS9 fusions to two different RNA editing enzymes to make C->U or A->I (G) edits in an RNA target. Our technology will be reversible and tunable, and will increase the variety, efficiency and safety of gene editing technologies. Current CRISPR/Cas9-based DNA editing depends upon endogenous DNA repair mechanisms. Homology- directed repair with a supplied template is the goal, though much more frequently non-homologous end joining occurs, often resulting in insertion or deletion (indel) mutations. Very recently, a method was described for CRISPR/Cas9-guided C -> U editing by fusing a catalytically dead Cas9 (dCas9) to the RNA/DNA editing enzyme APOBEC11 . However even this advance requires DNA repair mechanisms to correct the base mismatch, and additional manipulations are required to prevent reversion to the original, unedited base. Further, like all other CRISPR-based genome editing methods, the edits are not reversible. It was recently shown that it is possible to use CRISPR/Cas9 to target RNA2,3; editing of RNA using dCAS9 fusions to RNA editing enzymes like APOBEC1 or ADARs4 (A -> I RNA editors, where the inosine (I) is read as a guanine) may provide a novel mechanism for efficient, reversible, and targeted gene editing.
To examine the ability of a dCAS9-APOBEC1 fusion to edit RNA, we have designed an eGFP reporter with an ACG replacing the normal AUG start codon (Figure 1A). A gRNA and PAMmer oligo (a DNA oligo complementary to the RNA sequence in the region upstream and including the PAM site, which is required for RNA targeting2) have also been designed to direct the gRNA-dCAS9-APOBEC1 complex to the RNA. APOBEC1 edits only single stranded (ss)RNA and ssDNA; in the case of DNA editing1, upon hybridization of the gRNA to the 3’ DNA strand, APOBEC1 edited the target base in the unpaired region of the opposing 5’ DNA strand. In our attempt to edit RNA, there is no opposing unpaired strand within the gRNA binding site. We will therefore need to extend the length of the linker between the dCAS9 and APOBEC1 such that APOBEC1 can access the ssRNA downstream of the gRNA binding region (Figure 1B).
Upstream sequences in the RNA should be protected from editing by the presence of the PAMmer. The previous DNA editing study was able to demonstrate editing of bases up to 21nt away from the PAM using extended linkers1, which should be a sufficient distance to allow editing of RNA sequence downstream of the gRNA binding site. Thus, when our RNA editing complex binds the RNA in the appropriate location and makes the target C-> U edit, the start codon is restored and eGFP will be produced.
In a similar manner, we have designed a system to tether the RNA-specific A-> I editing enzyme ADAR1 to dCAS9. In this case, using a GFP reporter with a start codon mutation (such as AUA) is not optimal, because ADAR1 may edit the A in position 1 in addition to the A in position 3, thus eliminating translation initiation. We therefore designed an eGFP fusion to a well-documented reporter of nonsense-mediated mRNA decay (NMD), the beta-globin minigene5. This beta-globin eGFP reporter (Figure 2A) will contain a premature termination codon (PTC) in the second exon, which will target this mRNA for destruction by NMD. gRNA and PAMmers have been designed as described above to position the target A nucleotide immediately downstream of the start of the gRNA binding region. Upon targeting of this mRNA by our gRNA-dCAS9-ADAR1 complex, the PTC will be edited from a stop codon (UAG) to a tryptophan (UIG), thus resulting in the translation of the mRNA and expression of eGFP (Figure 2B).
(PICTURE 2 HERE)
Figure 2: A reporter system for CRISPR/Cas-targeted RNA editing by ADAR1. A beta-globin-eGFP mRNA with a premature termination codon (PTC) before the final intron (A, top) will be spliced and exported, and in the cytoplasm will be degraded by NMD (A, bottom); upon editing by gRNA-dCas9-ADAR1 complex, the PTC will be eliminated resulting in translation of eGFP (B).
The future potential applications and implications of this project are nearly endless. Our project will make it possible to make specific, tunable, and reversible edits to any RNA transcript. In addition to repairing diseased mRNAs relevant to human pathologies, our technology can be used in basic research as a novel method of gene regulation. For instance, our CRISPR-targeted RNA editors could be used to temporarily disrupt miRNA binding sites, splice sites, RNA-binding protein sites, and/or RNA secondary structure. In summary, we believe that our proposal for the development of novel CRISPR-targeted RNA editors offers several significant benefits over existing CRISPR-based DNA editing: 1) our system does not rely at all on DNA repair mechanisms; 2) A -> I (G) editing will be possible in addition to C -> U edits, thus expanding the variety of possible edits; 3) RNA editors will be tunable by regulating the quantity or efficiency of the editing complexes; 4) RNA edits are reversible because there is no permanent edit to the genome.
References:
1. Komor AC et al. Nature. Epub ahead of print (2016). 2. O’Connell MR et al. Nature. 516:263-266 (2014). 3. Nelles DA et al. Cell. 165:488-496 (2016). 4. Wang IX et al. Cell Rep. 5:849-860 (2013). 5. Zhang J et al. RNA. 4(7):801-15 (1998).
