Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

Hirosawa, Moe; Fujita, Yoshihiko; Parr, Christian; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

doi:10.1093/nar/gkx309

Cited by 97 publications

(78 citation statements)

References 28 publications

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“…A subsequent study by Hirohide Saito's group expanded this approach to further miRNA candidates (miR-21 and miR-302a) (27). Moreover, they added a negative feedback loop to the system, thereby establishing a positive relation between miRNA abundance and Cas9 activity (27). To this end, the authors expressed Cas9 from an mRNA harbouring an L7Ae binding motif (K-turn), while co-expressing the L7Ae repressor from an mRNA carrying miRNA binding sites in its 3'UTR (27).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, they added a negative feedback loop to the system, thereby establishing a positive relation between miRNA abundance and Cas9 activity (27). To this end, the authors expressed Cas9 from an mRNA harbouring an L7Ae binding motif (K-turn), while co-expressing the L7Ae repressor from an mRNA carrying miRNA binding sites in its 3'UTR (27). The resulting Cas-ON switch enabled miRNA-dependent Cas9 activation.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cell-specific CRISPR/Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

Hoffmann

Aschenbrenner

Große

et al. 2018

Preprint

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The rapid development of CRISPR/Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cell-specific CRISPR/Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

Hoffmann

Aschenbrenner

Große

et al. 2018

Preprint

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“…Hoffmann et al [106] developed a novel cell-type-specific Cas-ON switch based on AcrIIA4 and controlled by cellular miRNA signatures, confining spCas9 activity to hepatocytes or cardiomyocytes. In parallel with that work, Hirosawa et al constructed a synthetic mRNAdelivered Cas9-ON system using miRNA-responsive AcrIIA4 mRNA on the basis of improving the RNAbinding protein L7Ae-based miR-Cas9-ON switch they built previously [107,108]. Compared with a Cas-ON switch based on an L7Ae repressor, this Cas-ON switch design based on Acrs showed greater control than the previous system due to the high affinity of Acrs for Cas9-sgRNA complexes [106,107].…”

Section: Potential Application Of Type II Anti-crispr Proteinsmentioning

confidence: 99%

“…In parallel with that work, Hirosawa et al constructed a synthetic mRNAdelivered Cas9-ON system using miRNA-responsive AcrIIA4 mRNA on the basis of improving the RNAbinding protein L7Ae-based miR-Cas9-ON switch they built previously [107,108]. Compared with a Cas-ON switch based on an L7Ae repressor, this Cas-ON switch design based on Acrs showed greater control than the previous system due to the high affinity of Acrs for Cas9-sgRNA complexes [106,107]. Furthermore, a subsequent study validated the efficiency of this Cas-ON switch based on Acrs by adeno-associated virus vectors delivered to adult mice, and the data showed that NmeCas9, along with an AcrIIC3 constructed that was targeted for repression by liver-specific miR-122, allowed editing in the liver while repressing editing in heart muscle, indicating that this strategy provides safeguards against off-tissue genome editing [109].…”

Section: Potential Application Of Type II Anti-crispr Proteinsmentioning

confidence: 99%

Anti‐CRISPR proteins targeting the CRISPR‐Cas system enrich the toolkit for genetic engineering

2019

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Clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas adaptive immune defense systems, which are widely distributed in bacteria and Archaea, can provide sequence‐specific protection against foreign DNA or RNA in some cases. However, the evolution of defense systems in bacterial hosts did not lead to the elimination of phages, and some phages carry anti‐CRISPR genes that encode products that bind to the components mediating the defense mechanism and thus antagonize CRISPR‐Cas immune systems of bacteria. Given the extensive application of CRISPR‐Cas9 technologies in gene editing, in this review, we focus on the anti‐CRISPR proteins (Acrs) that inhibit CRISPR‐Cas systems for gene editing. We describe the discovery of Acrs in immune systems involving type I, II, and V CRISPR‐Cas immunity, discuss the potential function of Acrs in inactivating type II and V CRISPR‐Cas systems for gene editing and gene modulation, and provide an outlook on the development of important biotechnology tools for genetic engineering using Acrs.

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“…Temporal control can be achieved by smallmolecule induction of gRNAs 14,15 or Cas9, 16 but this comes with limitations in terms of multiplexing and spatial control. Spatiotemporal control has been achieved by regulation of Cas9 via photoactivation 17 or via tissue-specific promoters 18,19 or microRNAs, 20 which comes with the unwelcome restriction that all gRNAs are subject to the same regulatory scope. Systematic mapping of the structure and sequence properties of functional gRNAs has revealed that Cas9 activity is tolerant to significant modifications the standard gRNA structure, 21,22 facilitiating introduction of auxiliary domains that enable conditional control of gRNA activity via structural changes induced by small-molecules, 23,24 protein-bound RNAs, 25 nucleases, 26 or nuclease-recruiting DNAs.…”

Section: Introductionmentioning

confidence: 99%

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

Hanewich-Hollatz

Chen

Huang

et al. 2019

Preprint

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Dynamic RNA Nanotechnology in Living Cells Programmable RNA trigger X dCas9 Constitutively inactive (OFF ON logic) cgRNA Y Programmable DNA target Constitutively active (ON OFF logic) X dCas9 Y cgRNAA guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON→OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF→ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe programmable conditional gene silencing with a median dynamic range of ≈6-fold for an ON→OFF "terminator switch" mechanism, ≈15-fold for an ON→OFF "splinted switch" mechanism, and ≈3.6-fold for an OFF→ON "toehold switch" mechanism; the median crosstalk within each cgRNA library is <2%, <2%, and ≈20% for the three mechanisms. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

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Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

Cited by 97 publications

References 28 publications

Cell-specific CRISPR/Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

Cell-specific CRISPR/Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

Anti‐CRISPR proteins targeting the CRISPR‐Cas system enrich the toolkit for genetic engineering

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

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