Efficient genome editing using modified Cas9 proteins in zebrafish.

The zebrafish (Danio rerio) is an important model organism for basic as well as applied bio-medical research. One main advantage is its genetic tractability, which was greatly enhanced by the introduction of the CRISPR/Cas method a decade ago. The generation of loss-of-function alleles via the production of small insertions or deletions in the coding sequences of genes with CRISPR/Cas systems is now routinely achieved with high efficiency. The method is based on the error prone repair of precisely targeted DNA double strand breaks by non-homologous end joining (NHEJ) in the cell nucleus. However, editing the genome with base pair precision, by homology-directed repair (HDR), is by far less efficient and therefore often requires large-scale screening of potential carriers by labour intensive genotyping. Here we confirm that the Cas9 protein variant SpRY, with relaxed PAM requirement, can be used to target some sites in the zebrafish genome. In addition, we demonstrate that the incorporation of an artificial nuclear localisation signal (aNLS) into the Cas9 protein variants not only enhances the efficiency of gene knockout but also the frequency of HDR, thereby facilitating the efficient modification of single base pairs in the genome. Our protocols provide a guide for a cost-effective generation of versatile and potent Cas9 protein variants and efficient gene editing in zebrafish.

Cerebrospinal fluid cfDNA sequencing for classification of central nervous system glioma.

PURPOSE: Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained via surgery or biopsy, cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing (NGS) of CSF cfDNA in a representative cohort of 85 patients presenting at two neurooncological centers with suspicion of primary or recurrent glioma. Copy-number variation (CNV) profiles, single nucleotide variants (SNVs), and small insertions/ deletions (indels) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. RESULTS: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), non-malignant (n = 17) and non-diagnostic (n = 17). We introduced a molecular-guided tumor classification, which enabled identification of tumor entities and/ or cancer specific alterations in 75.0 % (n = 24) of glioblastoma and 52.6 % (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26-48 %) and SNVs at pre-defined gene loci (44 %), followed by SNVs/ indels identified via uninformed variant calling (8-14 %). A molecular-guided tumor classification was possible for 23.5 % (n = 4) of non-diagnostic cases. CONCLUSIONS: We developed a targeted sequencing workflow for CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma.