CRISPR-PIN: Modifying gene position in the nucleus via dCas9-mediated tethering

Abstract

Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair, genetic recombination, and gene expression. Here, we present CRISPR-PIN, a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae. This approach utilizes a cohesin-dockerin interaction between dCas9 and a perinuclear protein. In doing so, we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery. We demonstrate the utility of this approach for two applications: the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci. In both cases, we obtain results on par with prior reports using traditional, more cumbersome genetic systems. Thus, CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.

Keywords: CRISPR; Chromosome biology; Chromosome organization; Gene positioning; Synthetic biology.

Graphical abstract

Fig. 1

Development of CRISPR-dCas9-based gene positioning in the nucleus (CRISPR-PIN). (a) A schematic diagram showing the strategy to localize dCas9 (Streptococcus pyogenes) to the yeast nuclear periphery. Abbreviations: NPC, nuclear pore complex; Coh, cohesin; Doc, dockerin; and YFP, yellow fluorescent protein. (b) Confocal microscopy analysis nuclear localization of dCas9-Coh. Esc1 is a perinuclear membrane protein. Scale bar: 2 μm. (c) A schematic diagram showing the design of perinuclear organization of the acentric plasmid. Abbreviations: ARSH4, autonomous replicating sequence; TEF1p, TEF1 promoter; BFP, blue fluorescent protein; PRM9t, PRM9 terminator; LacO: lac operator. (d) Confocal microscopy analysis and quantification of nuclear localization of the acentric plasmid. The acentric plasmid localization is visualized via expression of GFP-LacI, which binds to the LacO array. (e) Quantification of localization is compared based on image analysis similar to (d). Abbreviations: LacI, lac repressor; and mCh, mCherry. n represents number of counted cells from three independent samples (n = 3), and data are shown in mean and standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch's independent 2-sample t-test. Scale bar: 2 μm.

Fig. 2

CRISPR-PIN aids the segregation of acentric plasmid. (a) A schematic diagram depicting the concept of the plasmid stability test. Cells are co-transformed with the acentric plasmids and CRISPR-PIN. A control plasmid containing a centromere undergoes normal segregation, whereas the acentric plasmid lacking a centromere fails to segregate properly. The presence of CRISPR-PIN with expression of gRNA targeting the plasmid assists the mitotic process, resulting in more stable acentric plasmid segregation. Abbreviation: CEN, centromere; gRNA, single guide RNA. (b) Quantification of plasmid stability. Data are collected by counting three synthetic dropout plates (n = 3), and presented in mean and standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA followed by pairwise Welch's independent 2-sample t-tests with Bonferroni correction. (c) Spot assay of cells co-transformed the acentric plasmids and CRISPR-PIN. 10-fold serial dilutions of overnight-grown cells are spotted on synthetic dropout plates. Growth was restored using the CRISPR-PIN approach to a level on par with the centromeric plasmid.

Fig. 3

CRISPR-PIN relocates the gene positions from various chromosomes. (a) The CRISPR-PIN approach is used to enable perinuclear localization of the target loci to the nuclear periphery. An array of TetO sites were integrated into the target loci for gene visualization. Abbreviation: TetO, Tet operator. (b) Representative fluorescence images showing perinuclear and nucleoplasmic localization of genes. Scale bar: 2 μm. (c) Quantification of nuclear localization of a series of genes. Chromosome number (Chr) of the gene is shown in Roman numerals. N represents number of counted cells from three independent samples (n = 3), and data are shown in mean and standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch's independent 2-sample t-tests between no gRNA controls and expressed gRNA targeted to each gene.