Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR.
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
Nature. 2017 Oct 25;
PubMed.
The novel feature of the Zhang system in the new Science paper is the use of a Cas protein/guide RNA complex for targeting specific RNA sequences. In other directed RNA-editing systems reported (i.e., from Joshua Rosenthal and Thorsten Stafforst labs), targeting specific sequences is accomplished simply by Watson-Crick pairing between a guide RNA and the target RNA, unassisted by any protein component. The Cas-driven recognition may provide another level to control affinity and selectivity, but this has yet to be fully evaluated or exploited. The Cas protein may also stabilize the guide RNA from cellular nucleases. One potential disadvantage of this system over other directed editing approaches that use endogenous ADARs is the need to deliver the large Cas protein (or its coding nucleic acid, which is also large). This is a particularly important consideration for therapeutic applications of directed RNA editing.
In general, directed editing of RNA (compared to DNA) has the advantage of being transient, allowing for temporal control of the target. Systems that create DNA mutations lead to permanent changes to the genome, which may not be desirable in all cases and, in fact, may be dangerous if the genomic editing reagent isn’t perfectly selective for its target site. All of the directed editing approaches reported (for RNA and DNA) have off-target sites. Future work to gather additional mechanistic and structural information on each system will allow for additional control of off-targeting. Indeed, Zhang shows here that detailed knowledge of the ADAR-RNA interface informed the design of specific mutants of his Cas13-ADAR fusion that had fewer off-target sites.
To change the message: RNA editing for neurodegenerative disorders
RNA has long been sought as a therapeutic target to provide transient amelioration of disease status without making permeant changes to the genetic markup. The recently reported RNA Editing for Programmable A-to-I Replacement (REPAIR) system is an ingenious combination of gRNA-mediated RNA-binding capability of a kinase domain-dead Cas13b and an adenosine deaminase to achieve single-base-pair RNA editing (a term coined way back at 1986). Such a system offers a highly efficient RNA editing tool to repair single-nucleotide mutations, so the bad message can be changed and “lost in translation.”
REPAIR is especially advantageous for studying pathogenesis and the development of gene therapy for neurodegenerative disorders. First, because the REPAIR system does not need to involve the endogenous DNA damage repair mechanisms, such as non-homologous end-joining or homology-directed repair, it provides more efficient editing in post-mitotic neurons. To study or combat neuroinflammation mediated by astrocytes and microglia, REPAIR offers the best scalpel due to its transient nature. Although the author only reported the A-to-I base-pair change, the system can be easily adapted to edit other bases, such as C to U changes via Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC). Furthermore, without the restraint of needing PAM sequence, REPAIR is flexible and can target any single-nucleotide mutation in the whole genome that associates with various familiar forms of neurodegenerative disorders. Finally, it can even be used to provide post-translational changes related to neurodegeneration by changing the phosphorylation target sites of the kinases.
One of the most obvious applications is to repair single-nucleotide mutations that lead to familiar forms of neurodegeneration. Such a system could also be harnessed to probe disease-associated mechanisms, e.g., to develop both cell-based and animal models of neurodegeneration. Furthermore, RNA-mediated neurodegeneration has been associated with Alzheimer's disease, Parkinson's disease, frontotemporal degeneration, amyotrophic lateral sclerosis, and Huntington’s disease. The mislocalization of RNA-binding proteins, microsatellite repeat-related generation of antisense transcripts and sequestration, repeat-associated non-ATG translation, aberrant mRNA splicing and processing, and noncoding RNA could all be potentially dissected and remedied by REPAIR.
Importantly, the transient nature of REPAIR may offer a way around the ethical debate about gene editing that causes permeant changes to the human genome. However, for therapeutic applications, there is still the remaining challenge of specificity and a potential for unwanted genetic modifications with an RNA-editing tool as described in the paper. Finally, adenosine-to-inosine (A-to-I) RNA editing regulated by neuronal excitation has been associated with normal neuronal function and neurodevelopment. ADAR2 is highly expressed in the brain. Mutations in ADAR2 have been related to epilepsy, seizures, autism and Fragile-X Syndrome, and neurodegeneration such as ALS, and the pathogenic mechanism is largely unknown. Therefore, even with the improved specificity in the REPAIR version 2, the potential consequence of the ectopic expression of kinase domain of the ADAR2 in perturbing the normal neuronal function needs to be further explored.
Comments
University of California, Davis
The novel feature of the Zhang system in the new Science paper is the use of a Cas protein/guide RNA complex for targeting specific RNA sequences. In other directed RNA-editing systems reported (i.e., from Joshua Rosenthal and Thorsten Stafforst labs), targeting specific sequences is accomplished simply by Watson-Crick pairing between a guide RNA and the target RNA, unassisted by any protein component. The Cas-driven recognition may provide another level to control affinity and selectivity, but this has yet to be fully evaluated or exploited. The Cas protein may also stabilize the guide RNA from cellular nucleases. One potential disadvantage of this system over other directed editing approaches that use endogenous ADARs is the need to deliver the large Cas protein (or its coding nucleic acid, which is also large). This is a particularly important consideration for therapeutic applications of directed RNA editing.
In general, directed editing of RNA (compared to DNA) has the advantage of being transient, allowing for temporal control of the target. Systems that create DNA mutations lead to permanent changes to the genome, which may not be desirable in all cases and, in fact, may be dangerous if the genomic editing reagent isn’t perfectly selective for its target site. All of the directed editing approaches reported (for RNA and DNA) have off-target sites. Future work to gather additional mechanistic and structural information on each system will allow for additional control of off-targeting. Indeed, Zhang shows here that detailed knowledge of the ADAR-RNA interface informed the design of specific mutants of his Cas13-ADAR fusion that had fewer off-target sites.
View all comments by Peter BealLSU Health Sciences Center
To change the message: RNA editing for neurodegenerative disorders
RNA has long been sought as a therapeutic target to provide transient amelioration of disease status without making permeant changes to the genetic markup. The recently reported RNA Editing for Programmable A-to-I Replacement (REPAIR) system is an ingenious combination of gRNA-mediated RNA-binding capability of a kinase domain-dead Cas13b and an adenosine deaminase to achieve single-base-pair RNA editing (a term coined way back at 1986). Such a system offers a highly efficient RNA editing tool to repair single-nucleotide mutations, so the bad message can be changed and “lost in translation.”
REPAIR is especially advantageous for studying pathogenesis and the development of gene therapy for neurodegenerative disorders. First, because the REPAIR system does not need to involve the endogenous DNA damage repair mechanisms, such as non-homologous end-joining or homology-directed repair, it provides more efficient editing in post-mitotic neurons. To study or combat neuroinflammation mediated by astrocytes and microglia, REPAIR offers the best scalpel due to its transient nature. Although the author only reported the A-to-I base-pair change, the system can be easily adapted to edit other bases, such as C to U changes via Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC). Furthermore, without the restraint of needing PAM sequence, REPAIR is flexible and can target any single-nucleotide mutation in the whole genome that associates with various familiar forms of neurodegenerative disorders. Finally, it can even be used to provide post-translational changes related to neurodegeneration by changing the phosphorylation target sites of the kinases.
One of the most obvious applications is to repair single-nucleotide mutations that lead to familiar forms of neurodegeneration. Such a system could also be harnessed to probe disease-associated mechanisms, e.g., to develop both cell-based and animal models of neurodegeneration. Furthermore, RNA-mediated neurodegeneration has been associated with Alzheimer's disease, Parkinson's disease, frontotemporal degeneration, amyotrophic lateral sclerosis, and Huntington’s disease. The mislocalization of RNA-binding proteins, microsatellite repeat-related generation of antisense transcripts and sequestration, repeat-associated non-ATG translation, aberrant mRNA splicing and processing, and noncoding RNA could all be potentially dissected and remedied by REPAIR.
Importantly, the transient nature of REPAIR may offer a way around the ethical debate about gene editing that causes permeant changes to the human genome. However, for therapeutic applications, there is still the remaining challenge of specificity and a potential for unwanted genetic modifications with an RNA-editing tool as described in the paper. Finally, adenosine-to-inosine (A-to-I) RNA editing regulated by neuronal excitation has been associated with normal neuronal function and neurodevelopment. ADAR2 is highly expressed in the brain. Mutations in ADAR2 have been related to epilepsy, seizures, autism and Fragile-X Syndrome, and neurodegeneration such as ALS, and the pathogenic mechanism is largely unknown. Therefore, even with the improved specificity in the REPAIR version 2, the potential consequence of the ectopic expression of kinase domain of the ADAR2 in perturbing the normal neuronal function needs to be further explored.
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