A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; peer review: 2 approved]

In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic ‘scars’. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions

A new inherited syndrome with severe neutropenia and neurological involvement due to autosomal recessive COPZ1 mutation

We identified a new homozygous stop-codon mutation in the COPZ1 gene (p.Q141X) in two siblings with severe neutropenia and neurological developmental delay. COPZ1 is a member of the coatomer protein complex I (COPI) regulating intracellular trafficking of proteins. CRISPR/Cas9-mediated introduction of the stop-codon mutation at the position p.Q141X in COPZ1 in healthy donors` cord blood hematopoietic stem cells (HSPCs) and iPSCs led to defective granulocytic differentiation in vitro. Additionally, copz1 mutant zebrafish embryos produced significantly fewer neutrophils than their control counterparts. These findings were in line with hyperactivated unfolded protein response (UPR) and elevated autophagy in the myeloid cell line NB4 after introduction of the truncated mutation in COPZ1. COPZ1 is ubiquitously expressed, while its paralogous gene, COPZ2, is absent in the blood and the brain. Interestingly, the rescue of COPZ1 mutated HSPCs with COPZ2 corrected the defective granulopoiesis. Thus, we describe a new severe congenital neutropenia syndrome caused by autosomal recessive COPZ1 mutations with downstream UPR and autophagy activation