Erratum: Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

[This corrects the article DOI: 10.1016/j.isci.2021.103463.].

Sizing, stabilising, and cloning repeat-expansions for gene targeting constructs.

Aberrant microsatellite repeat-expansions at specific loci within the human genome cause several distinct, heritable, and predominantly neurological, disorders. Creating models for these diseases poses a challenge, due to the instability of such repeats in bacterial vectors, especially with large repeat expansions. Designing constructs for more precise genome engineering projects, such as engineering knock-in mice, proves a greater challenge still, since these unstable repeats require numerous cloning steps in order to introduce homology arms or selection cassettes. Here, we report our efforts to clone a large hexanucleotide repeat in the C9orf72 gene, originating from within a BAC construct, derived from a C9orf72-ALS patient. We provide detailed methods for efficient repeat sizing and growth conditions in bacteria to facilitate repeat retention during growth and sub-culturing. We report that sub-cloning into a linear vector dramatically improves stability, but is dependent on the relative orientation of DNA replication through the repeat, consistent with previous studies. We envisage the findings presented here provide a relatively straightforward route to maintaining large-range microsatellite repeat-expansions, for efficient cloning into vectors.

Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.

Uses for humanised mouse models in precision medicine for neurodegenerative disease.

Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are very limited treatments, and no cures, for most of these diseases, including Alzheimer's Disease, Parkinson's Disease, Huntington Disease, and Motor Neuron Diseases. Mouse and other animal models provide hope by analysing them to understand pathogenic mechanisms, to identify drug targets, and to develop gene therapies and stem cell therapies. However, despite many decades of research, virtually no new treatments have reached the clinic. Increasingly, it is apparent that human heterogeneity within clinically defined neurodegenerative disorders, and between patients with the same genetic mutations, significantly impacts disease presentation and, potentially, therapeutic efficacy. Therefore, stratifying patients according to genetics, lifestyle, disease presentation, ethnicity, and other parameters may hold the key to bringing effective therapies from the bench to the clinic. Here, we discuss genetic and cellular humanised mouse models, and how they help in defining the genetic and environmental parameters associated with neurodegenerative disease, and so help in developing effective precision medicine strategies for future healthcare.

Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference.

Long non-coding RNAs (lncRNAs) have emerged as regulators of gene expression across metazoa. Interestingly, some lncRNAs function independently of their transcripts - the transcription of the lncRNA locus itself affects target genes. However, current methods of loss-of-function analysis are insufficient to address the role of lncRNA transcription from the transcript which has impeded analysis of their function. Using the minimal CRISPR interference (CRISPRi) system, we show that coexpression of the catalytically inactive Cas9 (dCas9) and guide RNAs targeting the endogenous roX locus in the Drosophila cells results in a robust and specific knockdown of roX1 and roX2 RNAs, thus eliminating the need for recruiting chromatin modifying proteins for effective gene silencing. Additionally, we find that the human and Drosophila codon optimized dCas9 genes are functional and show similar transcription repressive activity. Finally, we demonstrate that the minimal CRISPRi system suppresses roX transcription efficiently in vivo resulting in loss-of-function phenotype, thus validating the method for the first time in a multicelluar organism. Our analysis expands the genetic toolkit available for interrogating lncRNA function in situ and is adaptable for targeting multiple genes across model organisms.

Transcription driven somatic DNA methylation within the imprinted Gnas cluster.

Differential marking of genes in female and male gametes by DNA methylation is essential to genomic imprinting. In female gametes transcription traversing differentially methylated regions (DMRs) is a common requirement for de novo methylation at DMRs. At the imprinted Gnas cluster oocyte specific transcription of a protein-coding transcript, Nesp, is needed for methylation of two DMRs intragenic to Nesp, namely the Nespas-Gnasxl DMR and the Exon1A DMR, thereby enabling expression of the Gnas transcript and repression of the Gnasxl transcript. On the paternal allele, Nesp is repressed, the germline DMRs are unmethylated, Gnas is repressed and Gnasxl is expressed. Using mutant mouse models, we show that on the paternal allele, ectopic transcription of Nesp traversing the intragenic Exon1A DMR (which regulates Gnas expression) results in de novo methylation of the Exon1A DMR and de-repression of Gnas just as on the maternal allele. However, unlike the maternal allele, methylation on the mutant paternal allele occurs post-fertilisation, i.e. in somatic cells. This, to our knowledge is the first example of transcript/transcription driven DNA methylation of an intragenic CpG island, in somatic tissues, suggesting that transcription driven de novo methylation is not restricted to the germline in the mouse. Additionally, Gnasxl is repressed on a paternal chromosome on which Nesp is ectopically expressed. Thus, a paternally inherited Gnas cluster showing ectopic expression of Nesp is "maternalised" in terms of Gnasxl and Gnas expression. We show that these mice have a phenotype similar to mutants with two expressed doses of Gnas and none of Gnasxl.

Antisense Activity across the Nesp Promoter is Required for Nespas-Mediated Silencing in the Imprinted Gnas Cluster.

Macro long non-coding RNAs (lncRNAs) play major roles in gene silencing in inprinted gene clusters. Within the imprinted Gnas cluster, the paternally expressed Nespas lncRNA downregulates its sense counterpart Nesp. To explore the mechanism of action of Nespas, we generated two new knock-in alleles to truncate Nespas upstream and downstream of the Nesp promoter. We show that Nespas is essential for methylation of the Nesp differentially methylated region (DMR), but higher levels of Nespas are required for methylation than are needed for downregulation of Nesp. Although Nespas is transcribed for over 27 kb, only Nespas transcript/transcription across a 2.6 kb region that includes the Nesp promoter is necessary for methylation of the Nesp DMR. In both mutants, the levels of Nespas were extraordinarily high, due at least in part to increased stability, an effect not seen with other imprinted lncRNAs. However, even when levels were greatly raised, Nespas remained exclusively cis-acting. We propose Nespas regulates Nesp methylation and expression to ensure appropriate levels of expression of the protein coding transcripts Gnasxl and Gnas on the paternal chromosome. Thus, Nespas mediates paternal gene expression over the entire Gnas cluster via a single gene, Nesp.

Understanding functional miRNA-target interactions in vivo by site-specific genome engineering.

MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.

Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9.

We have applied the CRISPR/Cas9 system to Drosophila S2 cells to generate targeted genetic mutations in more than 85% of alleles. By targeting a constitutive exon of the AGO1 gene, we demonstrate homozygous mutation in up to 82% of cells, thereby allowing the study of genetic knockouts in a Drosophila cell line for the first time. We have shown that homologous gene targeting is possible at 1-4% efficiency using this system, allowing for the construction of defined insertions and deletions. We demonstrate that a 1 kb homology arm length is optimal for integration by homologous gene targeting, and demonstrate its efficacy by tagging the endogenous AGO1 protein. This technology enables controlled genetic manipulation in Drosophila cell lines, and its simplicity offers the opportunity to study cellular phenotypes genome-wide.

Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.

Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.

Identification and properties of 1,119 candidate lincRNA loci in the Drosophila melanogaster genome.

The functional repertoire of long intergenic noncoding RNA (lincRNA) molecules has begun to be elucidated in mammals. Determining the biological relevance and potential gene regulatory mechanisms of these enigmatic molecules would be expedited in a more tractable model organism, such as Drosophila melanogaster. To this end, we defined a set of 1,119 putative lincRNA genes in D. melanogaster using modENCODE whole transcriptome (RNA-seq) data. A large majority (1.1 of 1.3 Mb; 85%) of these bases were not previously reported by modENCODE as being transcribed. Significant selective constraint on the sequences of these loci predicts that virtually all have sustained functionality across the Drosophila clade. We observe biases in lincRNA genomic locations and expression profiles that are consistent with some of these lincRNAs being involved in the regulation of neighboring protein-coding genes with developmental functions. We identify lincRNAs that may be important in the developing nervous system and in male-specific organs, such as the testes. LincRNA loci were also identified whose positions, relative to nearby protein-coding loci, are equivalent between D. melanogaster and mouse. This study predicts that the genomes of not only vertebrates, such as mammals, but also an invertebrate (fruit fly) harbor large numbers of lincRNA loci. Our findings now permit exploitation of Drosophila genetics for the investigation of lincRNA mechanisms, including lincRNAs with potential functional analogues in mammals.