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

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 cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, 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 activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.

TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus.

Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys.

Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.

Impact of the Assembly-Activating Protein on Molecular Evolution of Synthetic Adeno-Associated Virus Capsids.

Over the last decade, the role of the assembly-activating protein (AAP) has begun to be dissected for the formation of adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, the authors' group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and AAP was found to be essential for capsid protein stabilization and generation of functional vector particles. Here, the lingering question is additionally addressed of whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame. To this end, a battery of complementary experiments was conducted in which: (1) the ability of chimeric AAP from AAVDJ, a hybrid of serotypes 2, 8, and 9, was tested to rescue AAP knockouts in the three parental serotypes; (2) the functionality of 60 chimeric AAPs extracted from five shuffled, unselected capsid libraries was measured; (3) whether production of different shuffled libraries, 10 wild-type serotypes or 25 individual chimeric capsids, can be enhanced by overexpression of AAP cocktails was assessed; and (4) the activity of 12 chimeric AAPs isolated from a shuffled library that was iteratively selected in vivo in mouse livers was studied. Collectively, the data demonstrate a remarkable tolerance of AAP for recombination via DNA family shuffling, evidenced by the findings that (1) all chimeric AAPs studied here retained at least partial activity, even in cases where the cognate hybrid capsid may be non-functional, and that (2) ectopic AAP overexpression did not enhance production of shuffled AAV chimeras or libraries, implying that the inherently encoded hybrid AAP variants are sufficiently active. Together, this work provides compelling evidence that AAP is not rate limiting during AAV capsid shuffling and thereby relieves a major concern in the field of AAV vector evolution.

A Robust and All-Inclusive Pipeline for Shuffling of Adeno-Associated Viruses.

Adeno-associated viruses (AAV) are attractive templates for engineering of synthetic gene delivery vectors. A particularly powerful technology for breeding of novel vectors with improved properties is DNA family shuffling, i.e., generation of chimeric capsids by homology-driven DNA recombination. Here, to make AAV DNA shuffling available to a wider community, we present a robust experimental and bioinformatical pipeline comprising: (i) standardized and partially codon-optimized plasmids carrying 12 different AAV capsid genes; (ii) a scalable protocol including troubleshooting guide for viral library production; and (iii) the freely available software SALANTO for comprehensive analysis of chimeric AAV DNA and protein sequences. Moreover, we describe a set of 12 premade and ready-to-use AAV libraries. Finally, we demonstrate the usefulness of DNA barcoding technology to trace AAV capsid libraries within a complex mixture. Our protocols and resources facilitate the implementation and tailoring of AAV evolution technology in any laboratory interested in customized viral gene transfer.

Blocking sense-strand activity improves potency, safety and specificity of anti-hepatitis B virus short hairpin RNA.

Hepatitis B virus (HBV) is a promising target for therapies based on RNA interference (RNAi) since it replicates via RNA transcripts that are vulnerable to RNAi silencing. Clinical translation of RNAi technology, however, requires improvements in potency, specificity and safety. To this end, we systematically compared different strategies to express anti-HBV short hairpin RNA (shRNA) in a pre-clinical immunocompetent hepatitis B mouse model. Using recombinant Adeno-associated virus (AAV) 8 vectors for delivery, we either (i) embedded the shRNA in an artificial mi(cro)RNA under a liver-specific promoter; (ii) co-expressed Argonaute-2, a rate-limiting cellular factor whose saturation with excess RNAi triggers can be toxic; or (iii) co-delivered a decoy ("TuD") directed against the shRNA sense strand to curb off-target gene regulation. Remarkably, all three strategies minimised adverse side effects as compared to a conventional shRNA vector that caused weight loss, liver damage and dysregulation of > 100 hepatic genes. Importantly, the novel AAV8 vector co-expressing anti-HBV shRNA and TuD outperformed all other strategies regarding efficiency and persistence of HBV knock-down, thus showing substantial promise for clinical translation.

CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans.

Engineering and evolution of synthetic adeno-associated virus (AAV) gene therapy vectors via DNA family shuffling.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties(1). These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors(2). A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol(1,2). Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling(3), i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically >80% for most AAV serotypes), or peptide display(4,5), i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.