Designing libraries for pooled CRISPR functional screens of long noncoding RNAs.

Human and other genomes encode tens of thousands of long noncoding RNAs (lncRNAs), the vast majority of which remain uncharacterised. High-throughput functional screening methods, notably those based on pooled CRISPR-Cas perturbations, promise to unlock the biological significance and biomedical potential of lncRNAs. Such screens are based on libraries of single guide RNAs (sgRNAs) whose design is critical for success. Few off-the-shelf libraries are presently available, and lncRNAs tend to have cell-type-specific expression profiles, meaning that library design remains in the hands of researchers. Here we introduce the topic of pooled CRISPR screens for lncRNAs and guide readers through the three key steps of library design: accurate annotation of transcript structures, curation of optimal candidate sets, and design of sgRNAs. This review is a starting point and reference for researchers seeking to design custom CRISPR screening libraries for lncRNAs

Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays

CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells

The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naive state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naive stem cells. Editing efficiencies of respectively 1.3-8.4% and 3.819% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naive hESCs were successfully differentiated to neural progenitor cells. As a proofof- principle of our workflow, two monoclonal genome-edited naive hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naive hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning

Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements

Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5′ adenine were improved by rescuing 5′ end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes

Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae

Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9technology, with its ability to target a specific DNA locus usingguide RNAs (gRNAs), is particularly suited for targeted mutagenesis.The targeted diversification of nucleotides in Saccharomycescerevisiae using a CRISPR-guided error-prone DNA polymerase calledyEvolvR was recently reported. Here, we investigate the effectof multiplexed expression of gRNAs flanking a short stretch of DNAon reversion and mutation frequencies using yEvolvR. Phenotypic assaysdemonstrate that higher reversion frequencies are observed when expressingmultiple gRNAs simultaneously. Next generation sequencing revealsa synergistic effect of multiple gRNAs on mutation frequencies, whichis more pronounced in a mutant with a partially defective DNA mismatchrepair system. Additionally, we characterize a galactose-inducibleyEvolvR, which enables temporal control of mutagenesis. This studydemonstrates that multiplex expression of gRNAs and induction of mutagenesisgreatly improves the capabilities of yEvolvR for generation of geneticlibraries in vivo

Parallel Genetics of Gene Regulatory Sequences in Caenorhabditis elegans

Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren.How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals

Transcriptome Analysis of Non‐Coding RNAs in Livestock Species: Elucidating the Ambiguity

The recent remarkable development of transcriptomics technologies, especially next generation sequencing technologies, allows deeper exploration of the hidden landscapes of complex traits and creates great opportunities to improve livestock productivity and welfare. Non-coding RNAs (ncRNAs), RNA molecules that are not translated into proteins, are key transcriptional regulators of health and production traits, thus, transcriptomics analyses of ncRNAs are important for a better understanding of the regulatory architecture of livestock phenotypes. In this chapter, we present an overview of common frameworks for generating and processing RNA sequence data to obtain ncRNA transcripts. Then, we review common approaches for analyzing ncRNA transcriptome data and present current state of the art methods for identification of ncRNAs and functional inference of identified ncRNAs, with emphasis on tools for livestock species. We also discuss future challenges and perspectives for ncRNA transcriptome data analysis in livestock species

In silico design and analysis of targeted genome editing with CRISPR

CRISPR/Cas systems have become a tool of choice for targeted genome engineering in recent years. Scientists around the world want to accelerate their research with the use of CRISPR/Cas systems, but are being slowed down by the need to understand the technology and computational steps needed for design and analysis. However, bioinformatics tools for the design and analysis of CRISPR experiments are being created to aid those scientists. For the design of CRISPR targeted genome editing experiments, CHOPCHOP has become one of the most cited and most used tools. After the initial publication of CHOPCHOP, our understanding of the CRISPR system underwent a scientific evolution. I therefore updated CHOPCHOP to accommodate the latest discoveries, such as designs for nickase and isoform targeting, machine learning algorithms for efficiency scoring and repair profile prediction, in addition to many others. On the other spectrum of genome engineering with CRISPR, there is a need for analysis of the data and validation of mutants. For the analysis of the CRISPR targeted genome editing experiments, I have created ampliCan, an R package that with the use of ‘editing aware’ alignment and automated normalization, performs precise estimation of editing efficiencies for thousands of CRISPR experiments. I have benchmarked ampliCan to display its strengths at handling a variety of editing indels, filtering out contaminant reads and performing HDR editing estimates. Both of these tools were developed with the idea that biologists without a deep understanding of CRISPR should be able to use them, and at the same time seasoned experts can adjust the settings for their purposes. I hope that these tools will facilitate adaptation of CRISPR systems for targeted genome editing and indirectly allow for great discoveries in the future