CRISPR 101: RNA Editing with Cas13 and REPAIR

Posted by Mary Gearing on Nov 30, 2017 9:01:02 AM


Cas13 enzymes are quickly becoming major players in the CRISPR field. Just a year after Abudayyeh et al. (2016) identified Cas13a (C2c2) as a RNA-targeting CRISPR enzyme, Cox et al. have adapted Cas13b for precise RNA editing. This new system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR tool for RNA editing, and it displays high specificity and targeting flexibility. We’ll walk through how this tool was developed and potential ways you can use it in your research.

Find the plasmids from Cox et al. here!

Cas13 Background and Advantages

Previous work established that Cas13a robustly cleaves RNA in bacteria, mammalian cells, and plant cells. Abudayyeh et al. (2017) compared knockdown with Cas13a from Leptotrichia wadei (LwaCas13a) to shRNA knockdown, finding that, like Cas9/Cpf1, Cas13a displayed higher specificity than shRNA-based systems. They also confirmed that catalytically inactive dCas13a could be used as a programmable RNA-binding protein and created the fluorescent fusion protein dLwaCas13a-NF for in vivo RNA imaging.

Cox et al. turned their attention to creating an RNA editing enzyme based on Cas13. RNA editing has multiple advantages over more traditional DNA editing systems; first, RNA editing doesn't require homology-directed repair (HDR) machinery, and could thus be used in non-dividing cells. Cas13 enzymes also don’t require a PAM sequence at the target locus, making them more flexible than Cas9/Cpf1. Some Cas13 enzymes prefer targets with a given single base protospacer flanking site (PFS) sequence, but orthologs like LwaCas13a do not require a specific PFS. Cas13 enzymes do not contain the RuvC and HNH domains responsible for DNA cleavage, so they cannot directly edit the genome. A Cas13-based RNA editing system would likely be reversible and would avoid genomic off-targets or indels introduced through non-homologous end joining (NHEJ).

Designing an RNA Editor

Cox et al. envisioned a two-component RNA editor: a Cas13 enzyme fused to an RNA adenosine deaminase (ADAR.) Such a system would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor would permit point mutations in RNA which could recapitulate or rescue known pathogenic alleles, or introduce a premature stop codon to render an RNA nonfunctional.

In their quest to build a robust RNA editor, Cox et al. started with the Cas13 scaffold, testing a whopping 21 Cas13a orthologs, 15 Cas13b orthologs, and 7 Cas13c orthologs. They hoped to find a stably folded ortholog that cleaved RNA robustly, unlike LwaCas13a, which must be stabilized by monomeric superfolded GFP and averages only ~50% RNA knockdown. In initial tests, Cas13b from Prevotella sep. P5-125 (PspCas13b) yielded 62.9% average knockdown, and they chose this enzyme for further studies. PspCas13b does not require a PFS, and it is sensitive to mismatches in target RNA from bases 12-26 of a 30 nt target sequence.

For the editing portion of the protein, Cox et al. examined ADAR1 and ADAR2, which deaminate adenosine to inosine in RNA, creating a functional A->G change. They fused ADAR deaminase domains (ADARDD) to dPspCas13b, but observed low RNA editing. To increase A->G editing, they employed hyperactive ADAR constructs, like ADAR2DD(E488Q). They also adjusted the structure of the guide RNA by introducing a C opposite the target A to be edited. This change specifies the edit to be made when multiple As are present in the gRNA spacer, as ADAR will preferentially edit an adenine if the template has a cytosine mismatch at that position.

CRISPR-RNA editing-horizontal.png

Cox et al. found that PspCas13b-ADAR2DD(E488Q) displayed robust editing with various spacer lengths from 30-84 nucleotides, and designated this system REPAIRv1. Using next-generation sequencing, they confirmed A->I editing and found that 50 nt spacers increase editing efficiency but also increase off-target editing within the target window, possibly due to longer stretches of duplexed RNA. Using an ADAR2DD catalytic mutant, they showed that editing is mediated by ADAR2DD, not PspCas13b.

Testing and Improving the Cas13b REPAIR System

To test the robustness of REPAIRv1, Cox et al. targeted 34 pathogenic G->A mutations from the ClinVar database. They successfully edited 33/34 sites with a maximum of 28% editing efficiency in HEK293T cells, as assessed using RNA-seq. Since the REPAIRv1 machinery is too large to fit into adeno-associated viral vectors, they tested truncated ADARDDs to see if they could shrink the construct. They identified ADAR2DD(delta984-1090), which decreases the REPAIRv1 construct size from 4,473 bp to 4,152 bp without decreasing editing efficiency.

Although REPAIRv1 knockdown is more precise than shRNA knockdown, it still displays substantial off-target activity. Cox et al. hypothesized that these off-targets were due to ADAR2DD, not Cas13b, and used protein engineering to optimize ADAR2DD. By mutating residues in ADAR2 that interact with duplex RNA, they hoped to destabilize ADAR-RNA binding to decrease off-targets. Of the tested constructs, ADAR2DD(E488Q/T375G) (REPAIRv2) had the highest on-target efficiency as well as the lowest amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target sites from the 18,385 observed with REPAIRv1 to merely 20.

Future Directions

Building on previous RNA targeting work with Cas13, REPAIR is the first CRISPR-based system to enable precise RNA editing. Cox et al. suggest potential avenues for creating new RNA base editors, including fusing dCas13b to a cytidine deaminase like APOBEC1 or engineering ADAR to accept cytidine substrates. As discussed above, the ability to edit RNA has multiple advantages, including reversibility and use in non-dividing cells.

Now that these REPAIR constructs have been shared with the scientific community, we look forward to learning more about their function across the entire transcriptome, and whether they can efficiently edit both low- and high-copy RNA species. We are also curious if REPAIR can function in a multiplexed manner to edit multiple RNAs, or multiple mutations in a single RNA. It’s clear that CRISPR RNA editors will open new doors for scientific research - how will you use this system in your research?


References

Cox, David B.T., et al. “RNA editing with CRISPR-Cas13.” Science (2017):pii: eaaq0180. PMID: 29070703.

Abudayyeh, Omar O., et al. “RNA targeting with CRISPR-Cas13.” Nature 550(7675) (2017):280-284. PMID: 28976959.

Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353(6299) (2016):aaf5573. PubMed PMID: 27256883. PubMed Central PMCID: PMC5127784.

East-Seletsky, Alexandra, et al. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection." Nature 538(7624) (2016):270-273. PubMed PMID: 27669025.

Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321. PubMed PMID: 28408723. PubMed Central PMCID: PMC5526198.

East-Seletsky, Alexandra, et al. "RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes." Mol Cell. 66(3) (2017):373-83. PubMed PMID: 28475872.

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