Eukaryotic-driven directed evolution of Cas9 nucleases.

BACKGROUND: Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity. RESULTS: Here, we develop a Eukaryotic Platform to Improve Cas Activity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity. CONCLUSIONS: We report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.

Combinatorial activation of the WNT-dependent fibrogenic program by distinct complement subunits in dystrophic muscle.

Fibrosis is associated with compromised muscle functionality in Duchenne muscular dystrophy (DMD). We report observations with tissues from dystrophic patients and mice supporting a model to explain fibrosis in DMD, which relies on the crosstalk between the complement and the WNT signaling pathways and the functional interactions of two cellular types. Fibro-adipogenic progenitors and macrophages, which populate the inflamed dystrophic muscles, act as a combinatorial source of WNT activity by secreting distinct subunits of the C1 complement complex. The resulting aberrant activation of the WNT signaling in responsive cells, such as fibro-adipogenic progenitors, contributes to fibrosis. Indeed, pharmacological inhibition of the C1r/s subunits in a murine model of DMD mitigated the activation of the WNT signaling pathway, reduced the fibrogenic characteristics of the fibro-adipogenic progenitors, and ameliorated the dystrophic phenotype. These studies shed new light on the molecular and cellular mechanisms responsible for fibrosis in muscular dystrophy and open to new therapeutic strategies.

Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency.

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.

Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare multisystem genetic disorder which is caused by genetic defects involving the Nipped-B-like protein (NIPBL) gene in the majority of clinical cases (60-70%). Currently, there are no specific cures available for CdLS and clinical management is needed for life. Disease models are highly needed to find a cure. Among therapeutic possibilities are genome editing strategies based on CRISPR-Cas technology. METHODS: A comparative analysis was performed to test the most recent CRISPR-Cas technologies comprising base- and prime-editors which introduce modifications without DNA cleavages and compared with sequence substitution approaches through homology directed repair (HDR) induced by Cas9 nuclease activity. The HDR method that was found more efficient was applied to repair a CdLS-causing mutation in the NIPBL gene. Human-induced pluripotent stem cells (hiPSCs) derived from a CdLS patient carrying the c.5483G > A mutation in the NIPBL were modified through HDR to generate isogenic corrected clones. RESULTS: This study reports an efficient method to repair the NIPBL gene through HDR mediated by CRISPR-Cas and induced with a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. This sequence repair method allowed the generation of isogenic wild-type hiPSCs clones with regular karyotype and preserved pluripotency. CONCLUSIONS: CdLS cellular models were generated which will facilitate the investigation of the disease molecular determinants and the identification of therapeutic targets. In particular, the hiPSC-based cellular models offer the paramount advantage to study the tissue differentiation stages which are altered in the CdLS clinical development. Importantly, the hiPSCs that were generated are isogenic thus providing the most controlled experimental set up between wild-type and mutated conditions.

Reporter-Based Isolation of Developmental Myogenic Progenitors.

The formation and activity of mammalian tissues entail finely regulated processes, involving the concerted organization and interaction of multiple cell types. In recent years the prospective isolation of distinct progenitor and stem cell populations has become a powerful tool in the hands of developmental biologists and has rendered the investigation of their intrinsic properties possible. In this protocol, we describe how to purify progenitors with different lineage history and degree of differentiation from embryonic and fetal skeletal muscle by fluorescence-activated cell sorting (FACS). The approach takes advantage of a panel of murine strains expressing fluorescent reporter genes specifically in the myogenic progenitors. We provide a detailed description of the dissection procedures and of the enzymatic dissociation required to maximize the yield of mononucleated cells for subsequent FACS-based purification. The procedure takes ~6-7 h to complete and allows for the isolation and the subsequent molecular and phenotypic characterization of developmental myogenic progenitors.

Non-coding Y RNAs associate with early replicating euchromatin in concordance with the origin recognition complex.

Non-coding Y RNAs are essential for the initiation of chromosomal DNA replication in vertebrates, yet their association with chromatin during the cell cycle is not characterised. Here, we quantify human Y RNA levels in soluble and chromatin-associated intracellular fractions and investigate, topographically, their dynamic association with chromatin during the cell cycle. We find that, on average, about a million Y RNA molecules are present in the soluble fraction of a proliferating cell, and 5-10-fold less are in association with chromatin. These levels decrease substantially during quiescence. No significant differences are apparent between cancer and non-cancer cell lines. Y RNAs associate with euchromatin throughout the cell cycle. Their levels are 2-4-fold higher in S phase than in G1 phase or mitosis. Y RNAs are not detectable at active DNA replication foci, and re-associate with replicated euchromatin during mid and late S phase. The dynamics and sites of Y1 RNA association with chromatin are in concordance with those of the origin recognition complex (ORC). Our data therefore suggest a functional role of Y RNAs in a common pathway with ORC.

Structural and functional analysis of four non-coding Y RNAs from Chinese hamster cells: identification, molecular dynamics simulations and DNA replication initiation assays.

BACKGROUND: The genes coding for Y RNAs are evolutionarily conserved in vertebrates. These non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate cells. However thus far, no information is available about Y RNAs in Chinese hamster cells, which have already been used to detect replication origins and alternative DNA structures around these sites. Here, we report the gene sequences and predicted structural characteristics of the Chinese hamster Y RNAs, and analyze their ability to support the initiation of chromosomal DNA replication in vitro. RESULTS: We identified DNA sequences in the Chinese hamster genome of four Y RNAs (chY1, chY3, chY4 and chY5) with upstream promoter sequences, which are homologous to the four main types of vertebrate Y RNAs. The chY1, chY3 and chY5 genes were highly conserved with their vertebrate counterparts, whilst the chY4 gene showed a relatively high degree of diversification from the other vertebrate Y4 genes. Molecular dynamics simulations suggest that chY4 RNA is structurally stable despite its evolutionarily divergent predicted stem structure. Of the four Y RNA genes present in the hamster genome, we found that only the chY1 and chY3 RNA were strongly expressed in the Chinese hamster GMA32 cell line, while expression of the chY4 and chY5 RNA genes was five orders of magnitude lower, suggesting that they may in fact not be expressed. We synthesized all four chY RNAs and showed that any of these four could support the initiation of DNA replication in an established human cell-free system. CONCLUSIONS: These data therefore establish that non-coding chY RNAs are stable structures and can substitute for human Y RNAs in a reconstituted cell-free DNA replication initiation system. The pattern of Y RNA expression and functionality is consistent with Y RNAs of other rodents, including mouse and rat.

Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication.

Non-coding Y RNAs are required for the initiation of chromosomal DNA replication in mammalian cells. It is unknown how they perform this function or if they associate with a nuclear structure during DNA replication. Here, we investigate the association of Y RNAs with chromatin and their interaction with replication proteins during DNA replication in a human cell-free system. Our results show that fluorescently labelled Y RNAs associate with unreplicated euchromatin in late G1 phase cell nuclei before the initiation of DNA replication. Following initiation, Y RNAs are displaced locally from nascent and replicated DNA present in replication foci. In intact human cells, a substantial fraction of endogenous Y RNAs are associated with G1 phase nuclei, but not with G2 phase nuclei. Y RNAs interact and colocalise with the origin recognition complex (ORC), the pre-replication complex (pre-RC) protein Cdt1, and other proteins implicated in the initiation of DNA replication. These data support a molecular 'catch and release' mechanism for Y RNA function during the initiation of chromosomal DNA replication, which is consistent with Y RNAs acting as replication licensing factors.