In the fission yeast the prevailing approach for Schizosaccharomyces pombe 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 (nourseothricin) selection marker. We also provide a web-tool, natMX6 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 cells, which S. pombe 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.
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.
In the fission yeast the prevailing approach for Schizosaccharomyces pombe 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 (nourseothricin) selection marker. We also provide a web-tool, natMX6 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 cells, which S. pombe 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.
Amendments from Version 1We have edited or added details in several places throughout the text to clarify the message. We have included a new relevant reference by Fernandez and Berro. We provide updated versions of Figure 2, Figure 4 and Figure 5 to improve data presentation, and provide a new section C for Figure 6 to show the chromosomal locations of the deleted non-coding RNA genes. We also provide a new Supplemental Figure 2 which contains the data used for the graphs in Figure 6A and B.
Para los mayas, a diferencia de Occidente, la matemática tiene un significado que sobrepasa los numerales y las operaciones entre ellos. Según su cosmovisión, la Deidad, la creadora del universo, es soberana en cuanto al cálculo matemático (Cabrera, 1992). La matemática maya ofrece la posibilidad de realizar las operaciones aritméticas de suma, resta, multiplicación, división y cálculo de raíces de forma sencilla, dado que la cultura maya logró sintetizar la cantidad mediante tres símbolos y logró sintetizar el valor con el sistema posicional en base veinte (Calderón, 1966). Se pretende en este artículo hacer un reconocimiento de los numerales mayas, su concepción metafórica y algunas operaciones aritméticas.
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