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

RESEARCH ARTICLE Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) Meri I. E. Uusi-Ma ¨kela ¨ 1 *, Harlan R. Barker 2 , Carina A. Ba ¨uerlein 1 , Tomi Ha ¨kkinen 3 , Matti Nykter 3 , Mika Ra ¨met 1,4,5 * 1Laboratory of Experimental Immunology, BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland, 2Laboratory of Anatomy, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland, 3Laboratory of Computational Biology, BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland, 4PEDEGO Research Unit, Medical Research Center Oulu, and Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland, 5Department of Pediatrics, Tampere University Hospital, Tampere, Finland *[email protected] (MR); meri.uusi-m[email protected] (MIEUM) Abstract 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 (Danio rerio) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity in vivo. Furthermore, some guide RNAs with high in vitro activity possessed poor mutagenesis activity in vivo, suggesting the presence of factors that limit the mutagenesis in vivo. 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. Introduction Since its discovery in Streptococcus pyogenes, the CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats–CRISPR associated 9) system has been extensively applied to modify the eukaryotic genome in a targeted manner [1,2]. CRISPR-Cas9 technology takes advantage of the bacterial Cas9 endonuclease, which generates a double stranded break in its DNA target [1]. The repair of the break by the error prone repair machinery of nonhomologous end joining often leads to the incorporation of mutations and permanent modifications to the genome [2]. PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 1 / 15 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Uusi-Ma¨kela¨MIE, Barker HR, Ba¨uerlein CA, Ha¨kkinen T, Nykter M, Ra¨met M (2018) Chromatin accessibility is associated with CRISPRCas9 efficiency in the zebrafish (Danio rerio). PLoS ONE 13(4): e0196238. https://doi.org/10.1371/ journal.pone.0196238 Editor: Bruce B. Riley, Texas A&M University, UNITED STATES Received: February 2, 2018 Accepted: March 9, 2018 Published: April 23, 2018 Copyright: ©2018 Uusi-Ma¨kela¨et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant experimental data are within the paper and its Supporting Information files. All files are available from the CRISPRz and the ArrayExpress databases (accession codes E-GEOD-45706; E-GEOD-4863; E-GEOD-52110; E-GEOD-74231). Funding: This study was supported with the following grants: Tampere Tuberculosis Foundation (http://www.tuberkuloosisaatio.fi/) (MU;MR); Finnish Concordia Fund (http://www.konkordialiitto.com/) (MU); Sigrid Juselius Foundation Cas9 is directed to bind its target sequence by a single chimeric guide RNA molecule (sgRNA), which recognizes an approximately 20 nucleotide target site, followed by the three nucleotide protospacer adjacent motif (PAM)-sequence (5’-NGG-3’) [1–3]. The sgRNA sequence is considered the limiting step in mutagenesis design, as the genomic target site needs to be unique. An optimal GC-content and specific nucleotides at key positions in the target sequence can also alter the efficiency and the specificity of mutagenesis [4–8]. The efficiency and unspecific, off-target binding of the nuclease are not easy to predict. As a result, multiple algorithms and online tools have been created for the identification of guide RNA targets with optimal Cas9 loading scores and the least amount of off-targets [9–17]. However, the in silico predictions do not always correlate with the observed mutagenesis efficiency and specificity [11,18–20]. Eukaryotic gene expression is regulated at the epigenetic level by packing of DNA into nucleosomes, which are formed by wrapping 146bp of DNA around a histone octamer [21]. These eukaryotic chromatin structures fundamentally differ from bacterial DNA packing, and being a prokaryotic enzyme, it is plausible that Cas9 cannot fully operate around all chromatin structures. Indeed, recent evidence indicates that chromatin influences Cas9 binding by limiting the accessibility of the target site [10,18,22–25]. Cas9 takes longer to scan for the target sites buried in heterochromatin, whereas targets located in euchromatin are more accessible, and thus easier to locate [24]. However, heterochromatin does not entirely prevent Cas9 from binding to potential target sites and despite binding, cleavage does not necessarily occur [22,24]. Target site accessibility is reflected in the tendency of Cas9 to act on secondary targets, so it plays an important role when designing effective sgRNAs with maximum efficiency and a minimal number of off-targets [10,17,18]. If the intended target is buried in heterochromatin, it is more probable that Cas9 binds to secondary targets and is more likely to find those in the exon regions in euchromatin [18]. Evidence supporting the involvement of chromatin accessibility in Cas9 binding has emerged in in vitro models, cell lines and in the zebrafish (Danio rerio) [10,17,23–26]. However, detailed understanding on which chromatin features contribute to chromatin accessibility this is still lacking. Compared to cell lines, zebrafish can present additional challenges for genome editing. Compared to other vertebrates, the teleost specific genome duplication has resulted in multiple similar genes or pseudogenes and this can, in some instances, complicate the identification of unique targets for sgRNA. Secondly, to generate mutant zebrafish, the sgRNA and Cas9 are microinjected into the fertilized embryo, and mutagenesis occurs during the first hours of development [27]. Compared to cell lines, the fertilized, CRISPR-injected zygote presents a challenge for all mutagenesis techniques as it undergoes developmental and differentiation processes that require global changes in chromatin. Lastly, the first cell division in zebrafish takes place very rapidly (40 minutes after fertilization), when compared to the cell divisions for example in mice (reaching E1.5 at 24 hours post fertilization, hpf). Mutagenesis occuring after this first cell division may more likely lead to mosaicism. During development, the chromatin landscape is under constant change in order to enable coordinated growth and differentiation [28–30]. The zygote is supported by the available maternal transcripts and the zygotic genome remains transcriptionally inactive until the maternal to zygotic genome activation (MZT) at the mid blastula transition (MBT) [31]. Our current understanding of zygotic chromatin is limited, but it has been shown that a specific histone modification pre-patterning marks developmentally active and inactive genes during development [32]. The nuclease accessibility of the developing, chromatin-packed genome of embryos remains poorly understood. Previously, it was observed that chromatin does not influence CRISPR-Cas9 targeting in zebrafish embryos in an MNase assay (Micrococcal nuclease assay), but later ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) results suggested that CRISPR-Cas9 is more likely to be successful when targeting open chromatin [9,17]. More Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 2 / 15 (http://sigridjuselius.fi/apurahat/) (MR); University of Tampere Doctoral School (http://www.uta.fi/ english/doctoralschool/index.html) (MU); Finnish Cultural Foundation – Maili Autio Fund (https://skr. fi/) (HB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. information on the influence of chromatin on CRISPR-Cas9 mutagenesis in model organisms is needed in order to improve the efficiency of genome engineering methodologies. In this study, we observed discrepancies between the in vitro and in vivo activities of sgRNAs, and that selected sgRNAs differ for their onset of mutagenesis. We saw an association between successful mutagenesis and the transcript levels during early development. We looked further into the involvement of gene activation and chromatin in explaining the CRISPR-Cas9 mutagenesis efficiency in zebrafish embryos. Our results indicate that gene expression and chromatin openness are associated with the efficiency of CRISPR-Cas9 mutagenesis. However, we saw no association of mutagenesis efficiency with either exon methylation or histone H3 Lysine 4 trimethylation (H3K4me3) at promoters. Results Good in vitro activity of sgRNA does not assure in vivo efficacy Analyzing the efficacy of different sgRNAs in vivo is laborious. To improve the screening for efficient sgRNAs, in vitro digestion of the target sequence can be used. We analyzed the mutagenesis activity of six sgRNAs first in vitro and then selected three for analysis in vivo. As shown in Fig 1, some sgRNAs with good in vitro efficiency presented low or no in vivo activity. This suggests that factors present in vivo prevent Cas9 from acting on its target site. Fig 1. In vitro and in vivo CRISPR-Cas9 mutagenesis efficiencies do not correlate for all genes. 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. https://doi.org/10.1371/journal.pone.0196238.g001 Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 3 / 15 Importantly, cxcr2 had neither detectable gene expression nor mutagenesis efficiency, whereas the genes permissive for mutagenesis (pycard,ca6) showed early expression (Fig 1,S1 Fig). This led us to hypothesize that the onset and the level of gene expression could influence the CRISPR-Cas9 mutagenesis. The corresponding results using the T7 Endonuclease I assay are displayed in S2 Fig. In our hands the T7 Endonuclease I assay has a lower resolution compared to the heteroduplex mobility assay, especially with sgRNAs of lower efficiency. On the other hand, the T7 Endonuclease I assay can be readily used for quantitation of mutagenesis efficiency, especially with sgRNAs of higher efficiency. The onset of mutagenesis differs between sgRNAs in vivo As we saw a discrepancy between in vivo and in vitro mutagenesis efficiencies for some sgRNAs, we next analyzed whether the onset of mutagenesis correlates with the onset of gene expression. To avoid the delay of mRNA transcription for Cas9 activity, we used a ready Cas9 protein in our experiments with appropriate preincubation step to allow the sgRNA to complex with Cas9. Three of our functional sgRNAs were chosen for the analysis. The sgRNAs targeting ca10a, sema4gb, or ca6 were co-injected with the Cas9 protein into the 1-cell stage embryo and the onset of mutagenesis was analyzed using both a heteroduplex mobility assay and a T7 Endonuclease I mutation detection assays. As shown in Fig 2 using the heteroduplex mobility assay, the first mutations become detectable as soon as 1hpf for ca10a and sema4gb, whereas the first mutations for ca6 appeared at 3hpf (Fig 2). These results indicate that the onset of mutagenesis differs depending on the sgRNAs in zebrafish embryos. Based on these results, we analyzed the relationship of early gene expression and mutagenesis efficiency in more detail with all our sgRNAs. We were able to detect mutagenesis activity at 1hpf (roughly corresponding to 4-cell stage). Likelihood of successful mutagenesis in relation to the expression level of the target gene in zebrafish embryos Altogether, we have designed 86 sgRNAs using the crispr.mit.edu, ChopChop (V1 and V2) and CRISPRscan softwares [9,14,15]. Of these sgRNAs, 30% showed detectable in vivo activity (S1 Fig 2. Onset of mutagenesis differs between sgRNAs. 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. https://doi.org/10.1371/journal.pone.0196238.g002 Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 4 / 15 Table). As GC-content (%) has been suggested to influence the effectiveness of CRISPR-Cas9 mutagenesis, we analyzed the GC-content of our sgRNAs (S1 Table) [18]. The GC-content of our functional sgRNAs was found to be similar (Mann-Whitney U-test; p-value 0.452) to that of the non-functional sgRNAs. When we compared the expression of the genes that we were able to mutate to those we were not, the genes resistant for mutagenesis more often had a very low expression level (Fig 3). However, the difference did not reach statistical significance (Fischer’s exact test; not significant). Moreover, a majority of genes (79%) permissive for mutagenesis underwent an increase in the number of transcripts around the MZT (identified here as a positive change in the number of transcripts between the oblong sphere stage and 50% epiboly). This occurred more often than in the genes resistant to mutagenesis (50%). However, this observation was not statistically significant (Fischer’s exact test) (Fig 3). To examine whether the lack of statistical significance was due to a type two error, we decided to determine whether there is a correlation between target gene expression and mutagenesis efficiency using larger datasets. Fig 3. Relationship of transcript levels in early development and low mutagenesis efficiency. Pie charts of the RNA-seq data corresponding to graphs in S1 Fig. 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 spherestage and 50% epiboly (around the MZT) (Fischer’s exact test; not significant). https://doi.org/10.1371/journal.pone.0196238.g003 Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 5 / 15 Mutagenesis efficiency correlates with gene expression and chromatin accessibility in zebrafish embryos In searching for factors that would explain the poor in vivo activity of some sgRNAs, we investigated available open access datasets. As genes with low expression values tended to be more difficult to mutate in our setting (Fig 3), we analyzed the association between expression levels and mutagenesis efficiency in greater depth, using large datasets in order to avoid type 2 error. We obtained CRISPR-Cas9 sgRNA efficiency data from CRISPRz database for all analyses [33]. We used open access RNA-seq data (E-GEOD-45706) for our primary analysis of the correlation between CRISPR-Cas9 mutagenesis and gene expression [33,34]. We found significant correlations in early development (between 64-cell stage and 36hpf), at the oblong sphere stage (3.66hpf, Spearman correlation 0.227; p-value 0.001) and at 36hpf (Spearman correlation 0.230; p-value 0.001). A strong correlation was observed at the oblong sphere stage which occurs shortly after MBT, around the time of zygotic genome activation. These results suggest that transcriptional activity influences CRISPR-Cas9 mutagenesis at early development (Table 1). As methylation is known to correlate with transcriptional repression, we used zebrafish exon methylation data to analyze whether there is any correlation between exon methylation and the success of CRISPR-Cas9 mutagenesis [33,35]. As is shown in Table 2, there was no significant correlation between exon methylation and CRISPR-Cas9 mutagenesis efficiency at the 1-cell stage or at MBT (Table 2). Similarly, using open access data on embryonic histone methylation, we analyzed whether there is a correlation of H3K4me3 at promoters with CRISPR-- Cas9 mutagenesis efficiency. As shown in Table 2, there seemed to be a correlation but this did not reach statistical significance (Spearman correlation 0.263; p-value = 0.074) [33,36]. ATAC-sequencing is a recent next generation sequencing method, which can be used to directly analyze chromatin accessibility. Open access ATAC-seq data for the zebrafish embryo is available at the 4hpf timepoint [37]. We compared mutagenesis efficiency data with ATACseq data at transcription start sites for a total of 263 genes. We discovered a significant, albeit rather weak correlation, indicating that chromatin accessibility appears to be one of the factors that explain the efficiency of CRISPR-Cas9 mutagenesis in zebrafish embryos (Table 2). Table 2. Correlation between mutagenesis efficiency and chromatin features at different developmental stages and timepoints. Chromatin feature n Developmental stage/Timepoint Spearman correlation p-value Exon methylation 263 1-cell 0.115 0.063 Mid blastula transition 0.107 0.084 H3K4me3 47 75–80% epiboly 0.263 0.074 Chromatin accessibility 263 4hpf 0.182 0.003 https://doi.org/10.1371/journal.pone.0196238.t002 Table 1. Correlation between mutagenesis efficiency and gene expression at different developmental stages during early development. n = 209. Developmental stage Spearman correlation p-value 64-cell 0.190 0.006 oblong-sphere 0.227 0.001 50%-epiboly 0.187 0.007 15-somite 0.210 0.002 36hpf 0.230 0.001 48hpf 0.182 0.008 60hpf 0.188 0.006 72hpf 0.131 0.058 https://doi.org/10.1371/journal.pone.0196238.t001 Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 6 / 15 Discussion In this study, we found discrepancies between the in vitro and in vivo efficiencies of some sgRNAs. These discrepancies suggested the presence of cellular factors which limit mutagenesis, and encouraged us to analyze chromatin involvement in more detail at the transcriptomic and epigenomic levels. Because the transcript counts of the early embryo can be masked by the presence of maternal transcripts, it is difficult to establish the exact relationship between gene expression and mutagenesis efficiency [38]. However, we found weak but significant correlations of gene expression with mutagenesis efficiency during early development, with the strongest correlation at the oblong sphere stage (3.66hpf, Spearman correlation 0.227; p-value 0.001) and later at 36hpf (Spearman correlation 0.230; p-value 0.001). The correlation at the oblong sphere stage suggests that genes which become active at the MZT are more accessible for Cas9 and hence undergo more efficient mutagenesis. As chromatin structure is complex, its effect on target site accessibility has to be determined for each structural level, starting with direct modifications to DNA bases, continuing with analysis of histone modifications signaling for open chromatin, and ending with analysis of chromatin accessibility. A detailed analysis is required in order to understand how CRISPR-- Cas9 mutagenesis activity could be manipulated at molecular level using for example chemical inhibitors of histone deacetylase activity. DNA methylation is known to mark transcriptional inactivity and recruit modified histones at the exons [39]. In our study, exon methylation was not found to significantly influence the activity of mutagenesis in zebrafish embryos. In confirmation, it has previously been suggested that Cas9 can act independently from DNA methylation in cell lines, and that, in general, most protein-DNA interactions are independent of DNA methylation [7,40]. If DNA methylation is not a limiting factor, we hypothesized that mutagenesis might correlate with higher order structures, specifically histone modifications. Various histone modifications mediate transcriptional activation and repression, and form nucleosome structures, which bind chromatin into an inactive heterochromatin state. H3K4me3 is a well known modification occurring in early development [41]. The most strongly suggestive, albeit not significant, correlation between experimental data and CRISPR-Cas9 mutagenesis efficiency was found with H3K4me3 data (Spearman correlation 0.263, p-value 0.07) [32]. This was expected, given the association with transcriptional activity at early developmental stages. As mutagenesis can already be detected at 1hpf it is possible that we fail to see a stronger correlation because the inspected timepoint is late and the histone landscape at 75–80% epiboly is dissimilar to that which is present before the MBT. In addition, if data from multiple timepoints would be available it would provide a more comprehensive view to opening of local chromatin structures. Also, observing only H3K4me3 signals might not accurately reflect the chromatin state in early embryos, as there are also other histone marks for open and closed chromatin, including H3K9me3 and H3K27me3 as well as H3K27ac at promoters [28,32,42]. A wider scale analysis of histone modifications could provide more insight into the association of CRISPR-Cas9 efficiency with histone landscape. A higher order structure above the histone landscape is shaped by modified histones organizing into nucleosomes. Nucleosome occupancy, breathing and remodeling have previously been found to affect the cleavage activity of Cas9 and consequently, CRISPR-Cas9 mutagenesis is more successful when targeting the sequences depleted in nucleosomes [25,26,43]. The position of the PAM-sequence relative to nucleosomes has been found to be a key determinant of the Cas9 endonuclease activity in vitro but not in zebrafish [17,23]. Nucleosomes affect chromatin accessibility, which can be measured using ATAC-seq [44]. This state-of-the-art method has been used for identification of accessible chromatin regions during early development [37]. Using the publicly available data, we found a weak but significant correlation between Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 7 / 15 chromatin accessibility and mutagenesis efficiency at the MBT, indicating that chromatin influences the efficiency of CRISPR-Cas9 mutagenesis in zebrafish embryos, even though it is not the sole defining factor (Table 2) [37]. Our results are in line with those by others [17] with different analysis method and dataset. Moreover, our results suggest CRISPR-Cas9 mutagenesis efficiency to be independent of exon methylation and H3K4me3 at promoters. Deciphering the effect of developmental chromatin on the activity of CRISPR-Cas9 mutagenesis model organisms ultimately leads us to an unanswered question about the regulation of zygotic genome activation and the signals that regulate this event at early stages before the MTZ [45]. The genome remains in a transcriptionally inactive state before the MZT, and it is likely that this inactive chromatin also limits the access of mutagenesis reagents such as Cas9. It is also likely that Cas9 can gain access during replication, and at sites that contain more permissive histone modifications or are depleted in nucleosomes, but only with limited efficacy. With further cell divisions, chromatin repressive signals then become diluted, leading to chromatin opening at the MZT and initiation of transcription [45]. Despite the biological significance of the MBT and MZT, we were already able to see mutagenesis taking place at 1hpf for some genes, so we propose that (when designing CRISPR-Cas9 mutagenesis strategies) chromatin structure should be taken into account at a very early timepoint (Fig 2). Several studies have looked into the correlation of in silico predictions and in vivo activity of sgRNAs and found that CRISPR-sgRNA design tools often fail to accurately predict sgRNA activity [11,20,25]. Moreover, it has been observed, that the in silico predictions which are efficient for model organisms are not efficient for cell line based assays and vice versa [11]. As Haeussler et al. (2016) observed, CRISPR-Cas9 efficiency in mice is better predicted by the algorithms that have been trained on zebrafish experimental data, than by cell line based algorithms. It is logical to assume this is at least in part due to the fact that mice and zebrafish undergo similar, conserved developmental dynamics at the transcriptomic and epigenomic level (at the time when CRISPR-mutagenesis is taking place), and target site accessibility is largely defined by early chromatin. Thankfully, design tools, which also take into account target site accessibility, have recently become available [11,14,16,17]. Detailed analysis is required to pinpoint which are the most important chromatin structures impacting CRISPR-Cas9 activity. With a better understanding of these, we will hopefully achieve improvements in predictions for experimental design especially in the in vivo models. Eventually, it might be possible to modify local chromatin to increase target site accessibility and simultaneously decrease the likelihood of off-target binding. Our results confirm the involvement of chromatin in defining CRISPR-Cas9 mutagenesis efficiency in a vertebrate model in vivo. Materials and methods Zebrafish maintenance Wild type AB fish were maintained in a flow-through system with a light/dark cycle of 14h/ 10h according to the standard procedure. Embryos and larvae were grown in an incubator (28.5˚C) in embryonic medium/E3 water (5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 , 0.33mM MgSO 4 , and 10–15% Methylene Blue). Ethics statement and data availability All experiments were carried out in accordance with the EU-directive 2010/ 63/EU on the protection of animals used for scientific purposes, and with the Finnish Act on the Protection of Animals Used for Scientific or Educational Purposes (497/2013) and the Government Decree on the Protection of Animals Used for Scientific or Educational Purposes (564/2013). We have only used zebrafish prior to their independently feeding larval stages in this study, which thus Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 8 / 15 do not require animal permits. Permit for the zebrafish housing and maintenance for the facility at the University of Tampere is ESAVI/10079/04.10.06/2015. The computational data analysed in this study were collected from open access sources, as detailed in the appropriate sections. Design and production of sgRNAs for CRISPR/Cas9 mediated genome editing Target sequences (S1 Table) for sgRNA design were chosen using the online based CRISPR design tool (http://crispr.mit.edu/), ChopChop.V1 or V2 [14,15] or CRISPRscan [9]. Target site uniqueness was verified with the NCBI BLAST analysis against the zebrafish genome (GRCz10). sgRNAs were produced as described previously [46]. Briefly, the sgRNA oligo (Sigma-Aldrich) and the T7 promoter site oligo (S1 and S2 Tables) (Sigma-Aldrich) were annealed and in vitro transcribed using the MEGAshortscript T7 Transcription Kit (Ambion Life Technologies, CA, USA). The integrity and size of the produced sgRNAs were analyzed with gel electrophoresis (1% agarose in Tris-acetate-EDTA, TAE). The concentration of the sgRNAs was measured with the Qubit1RNA BR Assay kit (Thermo Fisher Scientific, MA USA 02451) and Nanodrop 2000 (Thermo Fischer Scientific). sgRNA and Cas9 microinjection and genomic DNA extraction The sgRNAs and the Cas9 protein (ToolGen Inc., Seoul, South Korea) were co-injected into one-cell stage zebrafish embryos with a micro injector (PV830 Pneumatic PicoPump, World Precision Instruments) under a Nikon microscope (SMZ645), using borosilicate needles prepared with a Flaming/Brown micropipette puller. Needles were calibrated by injecting solution into a halocarbon oil droplet to achieve a diameter of 12μm (approximately 1nl). The embryos were aligned on 1.2% agarose E3 water plates prior to the injection. An injection solution containing 130ng/μl sgRNA and 250ng/μl of the Cas9 protein in nuclease-free water was incubated 37˚C 15min. Rhodamine dextran was added to the solution for the visualization of the injections under a Zeiss Lumar V12 fluorescence microscope. To analyze the onset of the mutagenesis 10–20 CRISPR-Cas9 injected embryos were collected and frozen in liquid nitrogen for DNA extractions at 1, 2, 3, 4, 6hpf (hours post fertilization). To analyze the in vivo mutagenesis efficiency, 5 embryos were collectedat 8hpf and immediately frozen in liquid nitrogen. For DNA extraction, the embryos were lysed 4h 55˚C in lysis buffer (10mM Tris pH 8,2, 10mM EDTA, 200mM NaCl, 0.5% SDS, 200μg/ml Proteinase K). DNA was precipitated 1h -20˚C using two volumes of ethanol. DNA was then pelleted by centrifuging 16,000g 10min. The pellet was washed with 200μl of 70% ethanol before resuspending in 200μl of water. A purification step with phenol-chloroform was performed after treatment with 15u of RNase A (Thermo Fischer Scientific) per 100μl of sample, 1h 37˚C. Heteroduplex mobility assay Targeted loci were amplified from the genomic DNA by PCR using the Maxima Hot Start DNA polymerase (Thermo Fischer Scientific) according to the manufacturer’s instructions. The PCR primers (S3 Table) were designed to anneal upstream and downstream of the expected cutting site. The PCR product was purified using Exo I and FastAP (Thermo Fischer Scientific) treatment 15min 37˚C, then 15min 85˚C. 10μl of the purified PCR product was annealed in a reaction containing 1x NEBuffer 2 (New England Biolabs, MA, USA) and was run on a 10% polyacrylamide gel. The gel was stained with GelRed (Bitium Inc., Fremont, CA). Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio) PLOS ONE | https://doi.org/10.1371/journal.pone.0196238 April 23, 2018 9 / 15