Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (<i>Danio rerio</i>)

<div><p>CRISPR-Cas9 technology is routinely applied for targeted mutagenesis in model organisms and cell lines. Recent studies indicate that the prokaryotic CRISPR-Cas9 system is affected by eukaryotic chromatin structures. Here, we show that the likelihood of successful mutagenesis correlates with transcript levels during early development in zebrafish (<i>Danio rerio</i>) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity <i>in vivo</i>. Furthermore, some guide RNAs with high <i>in vitro</i> activity possessed poor mutagenesis activity <i>in vivo</i>, suggesting the presence of factors that limit the mutagenesis <i>in vivo</i>. Using open access datasets generated from early developmental stages of the zebrafish, and guide RNAs selected from the CRISPRz database, we provide further evidence for an association between gene expression during early development and the success of CRISPR-Cas9 mutagenesis in zebrafish embryos. In order to further inspect the effect of chromatin on CRISPR-Cas9 mutagenesis, we analysed the relationship of selected chromatin features on CRISPR-Cas9 mutagenesis efficiency using publicly available data from zebrafish embryos. We found a correlation between chromatin openness and the efficiency of CRISPR-Cas9 mutagenesis. These results indicate that CRISPR-Cas9 mutagenesis is influenced by chromatin accessibility in zebrafish embryos.</p></div

Onset of mutagenesis differs between sgRNAs.

<p>Heteroduplex mobility assay to demonstrate the onset of mutagenesis using high efficiency guide RNAs targeting three different genes with different gene expression patterns in early development. Embryos were collected at timepoints 1, 2, 3, 4, 6hpf (15–20 embryos per group). The gene name above the gel image indicates CRISPR-Cas9 injected embryos and control indicates uninjected controls. The legend on the side indicates the positions of wt (wild type) and mutant bands in the gel. Red arrows indicate the point at which first mutations can be detected.</p

Correlation between mutagenesis efficiency and chromatin features at different developmental stages and timepoints.

<p>Correlation between mutagenesis efficiency and chromatin features at different developmental stages and timepoints.</p

Relationship of transcript levels in early development and low mutagenesis efficiency.

<p>Pie charts of the RNA-seq data corresponding to graphs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196238#pone.0196238.s004" target="_blank">S1 Fig</a>. a) Number of transcripts for the genes resistant to (left) or permissive (right) for mutagenesis between the oblong sphere and the 15-somite stage (Fischer’s exact test; not significant). 0.5 RPKM (Reads per Kilobase of transcript per Million mapped reads) was used as a limit for low expression. b) The number of genes resistant (left) or permissive (right) for CRISPR-Cas9 mutagenesis in which the number of transcripts is increased or decreased between the oblong sphere-stage and 50% epiboly (around the MZT) (Fischer’s exact test; not significant).</p

Correlation between mutagenesis efficiency and gene expression at different developmental stages during early development.

<p>n = 209.</p

In vitro and in vivo CRISPR-Cas9 mutagenesis efficiencies do not correlate for all genes.

<p>a) An in vitro digestion assay shows that sgRNAs differ in their efficiencies. Below the gene name, + and - indicate the presence or absence of Cas9 protein in the reaction. On the right the wild type (wt) and the mutant products are indicated. b) The in vivo CRISPR-Cas9 mutagenesis visualized for ca6, cxcr2 and pycard with a heteroduplex mobility assay, with the wild type (wt) and the mutant products indicated. 5 embryos were collected per sample at 8hpf.</p

Tumor detection by <i>in vivo</i> BLI correlates to M315 IgA serum measurement by ELISA.

<p>(<b>A</b>) M315 serum levels of melphalan treated (n = 5) and vehicle control (n = 5) on day 14 of treatment and simultaneous BLI measurement of the same mice. The treatment schedule is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052398#pone-0052398-g003" target="_blank">Figure 3A</a>. Measurement of idiotype specific M315 IgA significantly differed between the groups (two-tailed p value 0.0171) as it did with BLI (ventral+dorsal signal) (two-tailed p value 0.0221). (<b>B</b>) Scheme indicating time points for BLI measurement and serum collection for data presented in (C–G). (<b>C–G</b>) Idiotype specific M315 IgA serum levels of 5 untreated mice constantly increased over time correlating with progressing tumor burden as measured with BLI (ventral+dorsal signal). Of note, BLI measurements provided signals in early disease stages starting from day +9, whereas M315 IgA levels were not detectable at this time point. The left y-axis displays BLI signal intensity (black circles, solid lines); the right y-axis displays serum M315 IgA (red triangles, dashed lines).</p

Engraftment and growth dynamics of MOPC-315.BM luc⁺ myeloma cells in vivo.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. Tumor growth and spread was regularly monitored by BLI. (<b>A</b>) BLI images of one representative mouse at indicated time points after MM injection from ventral (top) and dorsal (bottom) view. Additional emerging tumor foci over time are marked with black or white arrows. (<b>B</b>) Number of skeletal spots per mouse on days +11 (n = 51), 19 (n = 56) and 33 (n = 25) and (<b>C</b>) percentage of mice presenting signals from liver and spleen. (<b>D</b>) Quantification of single tumor foci over time as marked in (A): 1 and 2 = BM compartment of femur/tibia, 3 = spleen, 4 = BM compartment of shoulder. (<b>E</b>) Absolute signal quantification by whole body BLI from ventral and dorsal views. (<b>F</b>) Representative osteolytic lesion in the neck of femur 42 days after MM injection. Corticalis is marked as c which is destroyed (arrow) by MOPC-315.BM luc⁺ cells marked with T. Original magnification 40X, scale bar is 200 µm. Insert: original magnification 200X, scale bar is 100 µm.</p

Detection of residing MOPC-315.BM luc⁺ cells and <i>in vivo</i> monitoring of reduced myeloma progression due to melphalan treatment.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. On day +19 after inoculation, tumors were established in all mice and readily detected by BLI. Then treatment was started ( = day 0 of treatment). Mice received 5 mg/kg melphalan (n = 9, two independent experiments) or mock treatment (vehicle control, n = 13, three independent experiments) intraperitoneally. One control group of MOPC-315.BM luc⁺ tumor bearing mice did not receive any treatment (untreated control, n = 14, three independent experiments). (<b>A</b>) Schematic study design, indicating treatment time points in respect to time after MM injection and end of experiment. Red arrows pinpoint melphalan treatment. (<b>B</b>) BLI images of two representative mice per group at selected time points in ventral (left) and dorsal (right) view. (<b>C</b>) Quantification of bioluminescence signal intensities over time from ventral or dorsal. Signals at day +19 were set as starting point and the following measurements were calculated as fold change of this initial signal intensity. Mice were treated at time points as indicated by arrows. Significant difference between melphalan treated mice vs vehicle control or vs untreated animals starting on day 10 of treatment for both, ventral (untreated vs melphalan p<0.0001, vehicle vs melphalan p = 0.0032) and dorsal (untreated vs melphalan p = 0.0006, vehicle vs melphalan p = 0.0024). (<b>D</b>) Quantification of skeletal tumor foci in untreated, vehicle control and melphalan treated mice on day 0 and 14 of drug treatment.</p

Flow cytometric measurement of surface receptors associated with BM homing and infiltration of myeloma cells.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. (<b>A</b>) 42 days after MM injection mice showed high BLI signals from hematopoietic compartments such as femur/tibia and spleen. Shown are two representative mice from ventral and dorsal view immediately before cells from BM and spleens were harvested for flow cytometry. (<b>B–D</b>) Besides BM and spleen derived MM cells, we also analysed MOPC-315.BM luc+ cells from culture. Dead cells were excluded by propidium iodide staining and MOPC cells identified as CD138⁺CD4⁺ double positive cells. (<b>B</b>) α4β1 integrin positive MOPC-315.BM luc⁺ cells were identified by flow cytometry as α4⁺ (CD49d⁺) and α4β7⁻. Representative FACS plots and the corresponding graph are shown, stating the frequency within CD138⁺CD4⁺ MOPC-315.BM luc⁺ cells expressing α4β1. For CXCR4 (<b>C</b>) and CD44 (<b>D</b>) representative histograms for each organ and cell line, including unstained fluorescence minus one (FMO) sample are displayed. Corresponding graphs state the fold difference in mean fluorescence intensity (MFI) related to the unstained FMO sample. BM and spleen: two independent experiments, n = 10, cells from cell culture: n = 4 for CXCR4 and CD44, n = 3 for α4β1. * indicates p<0.05 and ** indicates p<0.01 as determined by Kruskal-Wallis test with Dunn post test. (<b>E</b>) MOPC-315.BM luc⁺ cells were sorted for CXCR4^low and CXCR4^high expression. After 2 days in cell culture sorted cells regained the original CXCR4 expression level of the cell line. (<b>F</b>) 5×10⁵ sorted cells were i.v. injected into 4 female BALB/c mice each and BLI from ventral, lateral and dorsal was performed 10 days later. Sorted CXCR4^low as well as CXCR4^high CXCR4 cells readily homed to the BM compartment as well as to the spleen. (<b>G</b>) After BLI the mice were sacrificed, cells from left and right femur/tibia (separately) and the spleen extracted, and percentage as well as absolute numbers for CD138⁺CD4⁺ MM cells determined. From these values a ratio of spleen/BM was calculated to determine the homing capacity of the sorted populations. (<b>H</b>) Comparison of CXCR4 expression levels of sorted CXCR4^low and CXCR4^high cells immediately before injection and MM cells from BM and spleen after 10 days <i>in vivo</i> revealed a dynamic CXCR4 regulation.</p