Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Recent Advances in CRISPR Base Editing: From A to RNA Jennifer N. Bjerke,† Patrick C. Beardslee,† and Brian R. McNaughton*,†,‡ †
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, United States
‡
he flow of genetic information in living systems is encapsulated by the central dogma of molecular biology: DNA is transcribed into RNA, which is then translated into protein. In reality, of course, the story is much more complex and interesting. But fundamentally, this is us. Our DNA is constantly changing, as a result of (among other mechanisms) spontaneous deamination, photocycloadditions involving adjacent bases (thymine dimerization), or modification of a base with a reactive chemical. While we have DNA repair machinery to revert these changes throughout our genome, some persist. Relatively minute changes in our DNA can have no obvious affect at all or can lead to new and improved function(s) in encoded RNAs or proteins. However, in some cases, changes can lead to diseases like cancer. Given the relationship between DNA sequence and disease, a holy grail for much of the last half-century has been to chemically “fix”, with surgical precision, disease-relevant alterations to DNA, so-called gene editing. In addition to potential future therapeutic application, gene editing also allows researchers to reliably examine the relationship between a particular gene and disease. CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery consists of a Cas9 endonuclease and single-guide RNA (sgRNA). Once inside the nucleus of a cell, the Cas9−sgRNA complex selectively engages a DNA sequence complementary to the sgRNA, utilizing established rules for DNA−RNA base pairing. CRISPR-associated nucleases have been used for sequenceselective gene suppression by double-stranded DNA (dsDNA) cleavage and subsequent nonhomologous end joining (NHEJ). Additionally, CRISPR-associated nucleases have worked in concert with sequence-defined exogenous dsDNAs for precise gene editing through homology-directed repair (HDR). For a detailed discussion of the fascinating development of CRISPR, scholars are directed to recent perspectives.1,2 In the context of genome engineering, CRISPR-mediated dsDNA breaks can be risky business. Additionally, many diseases are the result of single-nucleotide polymorphisms (SNPs). These mutations are more rationally (and safely) addressed using “base editing”, which specifically reverts a SNP without inducing a dsDNA break and thus minimizes insertions and deletions. In seminal work, Liu and co-workers reported the first base editors,3 which convert G·C SNPs to A·T, and established a strategy reliant on targeted base modification followed by cellular mismatch repair to fix the complementary strand. More recently, the same laboratory has developed a CRISPRbased adenosine base editing (ABE) fusion protein to “fix” C·G to T·A mutations, which account for approximately half of all known pathogenic SNPs. In this system, a catalytically impaired Cas9 protein is fused to a deoxyadenosine deaminase enzyme.
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When complexed with a guide RNA, the complex engages a particular dsDNA sequence (dictated by the guide strand RNA) with an A·T SNP. The deoxyadenosine deaminase then converts the targeted adenine to inosine, which is subsequently converted to guanine following DNA replication and repair (Figure 1).4 At the outset of this research effort, there was one major problem: adenosine deaminase enzymes that target DNA do not exist in Nature! Using a tRNA adenine deaminase (TadA) as a starting point, Liu and co-workers developed a selection for base editing, which confers antibiotic resistance to bacteria expressing a Cas9−deoxyadenosine deaminase fusion protein capable of reversing a specific C·G to T·A mutation in an antibiotic resistance gene. After a herculean effort, involving many rounds of protein evolution and engineering, this team identified new adenine deaminase enzymes that reliably accept DNA as a substrate. In human cells, the most active ABEs reliably reverted specific disease-relevant C·G to T·A SNPs in γglobin (HBG1 and HBG2) and HFE genes. Principally, CRISPR has been used to modify DNA. However, there are benefits to editing RNA. For example, RNA editing potentially allows for temporal control, because mRNA is transient. For example, one could, in principle, stop editing the target RNA and rely on the transient lifetime of RNA and recurring transcription to eventually restore the original RNA sequence to the transcriptome pool. Recently, Zhang and co-workers reported an example of CRISPR-based RNA base editing, which they call RNA editing for programmable A to I replacement (REPAIR).5 This system relies on a fusion between a catalytically inactive CRISPR-associated RNAguided RNase (dCas13) and adenosine deaminase acting on RNA (ADAR) proteins. In concert with an RNA guide strand, the complex recognizes a target RNA. A specific cytidine in the guide RNA marks a mismatched adenosine for ADARdependent deamination to inosine, which can be functionally equivalent to guanosine in translation and splicing (Figure 2). However, the translational promiscuity (or lack thereof) of inosine at various positions in mRNA remains to be firmly established. Similar to the example described above, optimization of the fusion required extensive screening and protein engineering. First, a series of Cas13 proteins were assessed for sequence-specific mRNA targeting, in concert with a sequence complementary guide RNA, by measuring knockdown of mRNA that encodes a luciferase reporter protein. A catalytically inactive form of the most efficient Cas13 was fused to a deaminase domain of ADAR2 (ADAR2DD), which was able to essentially repair (by conversion of adenosine to inosine) a G → A nonsense mutation in a luciferase reporter. Subsequent Received: December 19, 2017
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DOI: 10.1021/acs.biochem.7b01276 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. A·T to G·C base editing (ABE). ABE is mediated by fusion between deoxyadenosine deaminase and catalytically impaired Cas9. Following DNA recognition, via an ABE-guide RNA−target DNA complex, a single-stranded DNA bubble is exposed that contains a targeted adenosine. By the action of an evolved deoxyadenosine deaminase, adenosine is converted into inosine. Following DNA repair or replication, the original targeted A·T base pair is replaced by a G·C base pair.
Figure 2. RNA editing for programmable A to I replacement (REPAIR). REPAIR is mediated by a fusion between catalytically impaired Cas13 b (dCas13b) and ADARDD, which deaminates adenosine to form insosine in dsRNA. A specific cytidine in the guide RNA marks a mismatched A for deamination to inosine, which mimics guanosine in translation and splicing.
protein engineering efforts were used to enhance adenosine deamination activity, as well decrease the level of off-target editing by introduction of mutations that destabilize ADAR2DD RNA binding. Using the optimized mutant, targeted REPAIR was achieved in multiple RNAs, with no observable off-target effects. As it sometimes goes, basic research can lead to scientific revolutions. In the case of CRISPR, basic questions about the dependency of salt concentration on restriction enzyme digestion of extremophile microbial DNA led to the identification of curious short regularly spaced repeats (SRSRs, an earlier-generation acronym). Subsequent fundamental questions about the biological role of these repeats revealed their use in adaptive immunity. Many additional studies illuminated the components of the system (e.g., Cas genes, guide RNA, protospacer, PAM, DNA target), which were soon programmed for targeted and reliable gene editing in various hosts. More recently, researchers have used protein engineering and evolution to expand the palette of CRISPRbased technologies. New developments in the laboratories of Liu and Zhang utilize catalytically inactive Cas proteins, and guide RNA, to selectively target DNA and RNA, respectively, and evolved or engineered fusions (deoxyadenosine deaminase or ADARDD) to selectively convert a specific adenine to inosine. Once installed, inosine is either converted to guanine (via DNA repair and replication) or read as guanine in RNA. These important papers expand the palette of CRISPR base editing and will likely find use by a diverse array of biomedical researchers to specifically “fix” DNA or RNA with an adenine point mutation, as well as study the role of specific point mutations (at the DNA or RNA level) in disease.
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Funding
B.R.M. acknowledges funding form the National Institutes of Health (R01 GM107520 and R01 GM123864). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Ledford, H. (2016) The unsung heroes of CRISPR. Nature 535, 342−344. (2) Lander, E. S. (2016) The Heroes of CRISPR. Cell 164, 18−28. (3) Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., and Liu, D. R. (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420−424. (4) Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., and Liu, D. R. (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464−471. (5) Cox, D. B. T., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., and Zhang, F. (2017) RNA editing with CRISPR-Cas13. Science 358, 1019−1027.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Brian R. McNaughton: 0000-0002-5002-2262 B
DOI: 10.1021/acs.biochem.7b01276 Biochemistry XXXX, XXX, XXX−XXX
