Expression and epigenetic profile of different CRISPR-Cas9 efficiency clones

CRISPR/Cas9 cleavage efficiency is crucial in a genomic editing experiment. However, the molecular mechanisms that underlie this cleavage difference remain unclear. In our study, we characterized genome-wide gene expression and epigenetic features in CRISPR-Cas9 low and high clones across 3 different cell lines. We show that Cas9 expression level is relatively higher in high efficiency clones. Notably, histone mark ChIP-seq data demonstrate that differential expressed genes also have a different acetylation of histone 3 at lysine 27 (H3K27ac) level. Finally, we observed that PARVA is an important gene that can enhance CRISPR-Cas9 efficiency.

chosen exon [7]. However, it is not clear what factors may affect the efficiency. So we did the expression and epigenetic profile in CRISPR-Cas9 high and low efficiency clones in order to illustrate the point.

Results
Cas9 single clone shows differential cleavage efficiency by T7E1 digestion examination We obtained different CRISPR/Cas9 cell lines from Wei lab. Among them, one is HeLa-sc8 high efficiency clone as they reported previously [5]. Other 2 cell lines are HT1080 and 293T. We used sgRNA targeting CSPG4 gene to validate the cutting efficiency as reported [8]. Different CRISPR editing efficiency clones are showed in gel figure (Fig 1a).
The efficiency ranges from 3% to 27%. Then we carried out a genome-wide transcriptomic and epigenetic assessment on these clones (Fig 1b). RNA-seq and H3K27ac ChIP-seq were performed respectively in these clones.

Exploring the genomic expression difference in CRSIPR-Cas9 high and low efficiency clones by RNA-seq
On average, we sequenced each sample at ~50 million PE50 fragments and observed 90% read mappability (Supplementary file 2). Cas9 with OCT1 and BSD were constantly expressed in different clones (Fig 2a). We performed RNA-seq in 293T, HT1080 and HeLa to seek the genes that may correlate with cleavage efficiency. RPKM value was calculated for Cas9, BSD and Oct1 respectively (Fig 2a). Gene expression analysis revealed that Cas9 is higher expressed in high efficiency clone than low efficiency clone (Fig 2b). OCT1 and BSD genes are also showing the same pattern with Cas9 (Fig 2b). A total of more than 200 genes were differentially expressed in high and low clones. Then we performed Gene Ontology analysis for differential expressed genes (Fig 2c). GO analysis showed that differential expressed genes are extracellular matrix organization, extracellular structure organization related genes (Fig 2c). Specifically, antigen processing and presentation of peptide antigen via MHC class-related genes were up-regulated in efficiency high clones in 293T cells. We next analyzed the genes that were differentially expressed between CRSIPR-Cas9 high efficiency clone and low efficiency clone. There are 58 up-regulated and 35 down-regulated genes in CRISPR/Cas9 high efficiency clones relative to low efficiency clones (Fig 2d and Supplementary file 1). In addition, UNC5B was up-regulated in high efficiency clones compare to low efficiency clones, and PARVA was down-regulated in high efficiency clones compare to low efficiency clones (Fig 2d).

Exploring the epigenetic difference of CRISPR/Cas9 high and low efficiency clones by H3K27ac ChIP-seq
Epigenetic changes are important features in biological process [9][10][11]. We next examined the acetylation of histone 3 at lysine 27(H3K27ac) level of CRISPR/Cas9 high and low efficiency clones. We performed H3K27ac ChIP-seq in 293T low and high CRISPR/Cas9 efficiency cell lines. H3K27ac, which is a marker of actively transcribed genes, showed clear differences between high and low efficiency clones. In total, 77,977 peaks were identified in high efficiency clones, and 77,606 peaks were identified in low efficiency clones. Venn diagram shows that H3K27ac peak overlap by number of 51,107 (Fig 3a). We then profiled H3K27ac signal on differential genes between 293T high efficiency clone and 293T low efficiency clones (Fig 3b). Differential expressed genes also have different H3K27ac level. We separated differential expressed genes into 2 groups, including up genes and down genes. For up genes, H3K27ac level is higher on the nearby peaks in CRISPR-Cas9 high efficiency clone than CRISPR-Cas9 low efficiency clone (Fig 3c, up   panel). For down genes, H3K27ac level is higher on the nearby peaks in CRISPR-Cas9 low efficiency clone than CRISPR-Cas9 high efficiency clone (Fig 3c, down panel). Genome browser showed unique peaks in low and high CRISPR-Cas9 efficiency cell lines (Fig 3d).
Integration of our epigenetic and transcriptome data showed that 120 genes were up regulated and marked with histone modifications in high efficiency cells, whereas 230 genes were down regulated across the 3 cell lines. Collectively, these results show that high efficiency clones display distinct gene expression and epigenetic changes compared with low efficiency clones.

Identification of the PARVA expression level in improving CRISPR-Cas9 cutting efficiency
We next seek the role of PARVA in improving CRISPR-Cas9 cutting efficiency. We built PARVA overexpression lentirirus vector and transfected CRISPR-Cas9 low efficiency clones.
3 days after transfection, cells are lysed and total RNA was harvested. RT-PCR shows that PARVA was overexpressed successfully (Fig 4a). We used sgRNA targeting CSPG4 gene to validate the cutting efficiency as reported [8]. The efficiency is enhanced from 3% to 27% by T7E1 digestion analysis (Fig 4b).
Discussion PARVA encodes a member of the parvin family of actin-binding proteins [12]. Parvins are associated with focal contacts and contain calpomi homology domains that bind to actin filaments. The encoded protein is part of the integrin-linked kinase signaling complex and plays a role in cell adhesion, motility and survival. The infection efficiency of adenovirus is determined by the levels of CAR and integrin on cancer cell [13]. That's why PARVA gene expression level can improve CRISPR-Cas9 cleavage efficiency. PARVA also promotes metastasis by modulating ILK signaling patheway in lung adenocarcinoma [14].

Conclusions
To improve CRISPR-Cas9 gene editing efficiency, we recommend taking Cas9 and PARVA expression level together into consideration.