Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish

Abstract

Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

Fig 1. CRISPR/Cas9 mainly generated mutation before dome stage in zebrafish.

gRNAs targeting on 4 genes (smc1a, tarbp1, vps13c, ccdc129) were injected into the one-cell embryos of zebrafish, and mutation events were detected by using restriction enzymes (RE) at following embryogenesis stages: 64-cell (2 hpf), 256-cell (2.5 hpf), 1k-cell (3 hpf), dome (4.33 hpf), bud (10 hpf), 24 hpf and 48 hpf, a 48 hpf control was included. (A) PCR products of each Cas9 target were treated by specific RE. (B) Fluorescence intensity was transferred to mutation rates by ImageJ. Blue, red, purple and grey line represent mutation rate of gRNAs targeting on smc1a, tarbp1, vps13c and ccdc129, respectively. X-axis was arranged according to hpf time course of zebrafish embryo development. Error bars were calculated from replicates of gRNAs targeting on each gene.

Fig 2. CRISPR/Cas9 efficiency did not show clear correlation with nucleosome organization.

(A) Schematic diagram of nucleosome organization status identification according to our previous MNase-seq data (GSE44269, see Material and Method section for details). Zebrafish genebody (Refseq annotation) was divided into nucleosome linker (NL, blue) region, nucleosome organization (NO, red) region, and dynamic nucleosome region (grey). (B) Distribution of mutation rate. (C) Barplot of proportion of effective gRNA targeting on NL region (left) and NO region (right), odds ratio: 1.22. (D) Boxplot of mutation rate of gRNA targeting on NL region (blue) and NO region (red), t-test p-value: 0.371.

Fig 3. CRISPR/Cas9 efficiency is positively correlated with chromatin accessibility.

(A) Schematic diagram of chromatin accessibility status identification according to our ATAC-seq data (see Material and Method section for details). Zebrafish genome was divided into open chromatin (OC, blue) region and close chromatin (CC, red) region. (B) Barplot of proportion of effective gRNA targeting on OC region (left) and CC region (right), odds ratio: 1.52. (C) Boxplot of mutation rate of gRNA targeting on OC region (blue) and CC region (red), t-test p-value: 0.0952.

Fig 4. Paired co-injection experiments indicated chromatin accessibility information could be applied to improve gRNA designs.

(A) Dot plot of mutation rates for gRNAs targeting on OC regions (blue), and gRNAs targeting on CC regions (red). (B) Barplot of the mutation rate differences between paired co-injected gRNAs. (C) Boxplot of mutation rates of gRNA targeting on OC region (blue) and CC region (red), paired t-test p-value: 0.0199. (D) Summary of discretized mutation rates for gRNAs targeting on OC regions and CC regions separately.

Fig 5. A webserver was developed to improve CRISPR/Cas9 target design.

(A) Cumulative proportion curve of number of genes (Refseq annotation) on which there are at least 1–100 potential gRNA targets on OC region (blue). 48.61% of genes have at least 5 targets on OC region. (B) Screenshot of a query example for gene smarca4 exon 1 on OC regions at the online webserver.