Homologous Recombination-Enhancing Factors Identified by Comparative Transcriptomic Analyses of Pluripotent Stem Cell of Human and Common Marmoset

A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination activity in marmoset ESCs. Here, we compared gene expression in marmoset and human pluripotent stem cells using transcriptomic and quantitative PCR analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D, and RAD51) were upregulated in marmoset cells. A total of four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.

The genome editing CRISPR-Cas9 technology [5] relies on endogenous pathways for repairing DSBs in cells [6]. Although the combination of CRISPR-Cas9 and double-stranded DNA (dsDNA) donor enables precisely targeted integration or deletion of long sequences by HDR, the efficiency of this process is limited by competing DSB repair pathways, mainly NHEJ. Previous studies have demonstrated that the efficiency of Cas9-meditated HDR can be modified through use of small molecules that stimulate the HDR-related factor RAD51 [7,8] or that inhibit the NHEJ-related factor LIG4 [9,10]. Additionally, modification of Cas9 by fusion with DSB repair-related factors such as RAD52, CtIP, MRE11A [11][12][13], or a dominant negative mutant of P53BP1 [14] can improve HDR efficiency. Moreover, overexpression of several DSB-related genes, including RAD52 [12] and RAD18 [15], reportedly contributed to enhancement of Cas9-mediated HDR efficiency.
For passaging, cells were pre-treated with 10 µM Rho-associated coiled-coil-containing protein kinase inhibitor Y-27632 (Wako, Tokyo, Japan) in ESM at 37 • C for an hour. The cells were then incubated in CTK solution (Reprocell) at 37 • C for 30 s, mechanically separated from feeder cells, and dissociated by gentle pipetting. The isolated cells were plated onto new feeder cells in ESM supplemented with 10 µM Y-27632. After twenty-four hours, Y-27632 was removed from the medium. Medium change was performed daily. Prior to the seeding of hESCs and iPSCs, feeder cells were seeded onto a gelatin-coated 100 mm cell culture dish in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% inactivated fetal bovine serum (Thermo Fisher Scientific).
For transfection (Day 0), we used the NEPA21 Super Electroporator (Nepagene) as described previously [20]. A total of ten µg of HPRT-TV and 5 µg each of the Cas9/gRNA and overexpression vectors were diluted in 100 µl of OPTI-MEM (Thermo Fisher Scientific). The etKA4 hiPSCs (>1 × 10 7 cells) were suspended in the solution and subjected to electroporation. Transfected cells were plated onto new mitomycin-C-treated G418resistant SNL76/7 feeder cells on a 100-mm cell culture dish in ESM supplemented with 10 µM Y-27632. On Day 2, selection was initiated by adding 100 ng/mL G418 (an analog of neomycin; Sigma) to the ESM; selection was performed for six days. On Day 8, the concentration of G418 was doubled, and selection was continued for an additional three days. On Day 11, the number of G418-resistant colonies was counted. The cells were then subjected to further selection in 10 µM 6-thioguanine (6TG) for five days. On Day 16, the number of 6TG-resistant colonies was counted. Experimental data were only included when at least 80 G418-resistant colonies survived.
For cell cycle analysis, we used the Fucci2.1 system [47] by lentiviral transfection of CSII-EF-mCherry-hCdt1 and CSII-EF-AmCyan-hGem to the etKA4 iPSCs, and one doublepositive clone was chosen and used for further transfection of no-DNA (termed as "sham") or the BRCA2 vector as described above. We used a FACSVerse (BD Biosciences, New Jersey, United States) for the cell cycle analysis, with 7-AAD (Thermo Fisher Scientific) for removing dead cells.

Genotyping
Southern blotting was performed as described previously [35] using the digoxigenin (DIG) probe system. Genomic DNA samples were digested with BglII and EcoRV by overnight 37 • C incubation. We used the PCR DIG Probe Synthesis Kit (Roche) for DIG-labeled probe production. The human HPRT-specific probe (492 bp) was amplified from human genomic DNA using the primers TGCATATCTGGGATGAACTCTGG and AAATGGGACATTTGTGTGTCACC. Molecular sizes were confirmed using DIG-labeled DNA Molecular Weight Marker II (λDNA with Hind III digestion) (Sigma; #11218590910). Genotyping PCR and DNA sequencing analysis ( Figure S1, S5 and S6) were performed as described previously [20].

Quantitative Reverse-Transcription PCR (qPCR)
RNA extraction, reverse transcription, and PCR were performed as described previously [35]. A total of three biological and technical repetitions of the qPCR analyses were performed. Quantification was performed using the relative standard curve method and endogenous expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primers were newly or previously designed to amplify both human and marmoset cDNA sequences; however, they did not amplify murine cDNA sequences to avoid contamination due to the use of MEFs for PSC culture [35,48]. The following primers were used: , and BRCA2-reverse (GTATACCAGCGAGCAGGCCG).

RNA-Seq Analysis
Transcriptome data were obtained from cmESCs as described previously (GSE138944) [49]. We used the deposited RNA-seq data from human ESCs and iPSCs (GSE53096) [50] as a reference. Marmoset mRNA was sequenced on an Illumina HiSeq2500 and the obtained nucleotide sequences were mapped against the Callithrix jacchus genome (Cal-lithrix_jacchus_cj1700_1.1; https://www.ncbi.nlm.nih.gov/assembly/GCF_009663435.1/ (accessed on 1 December 2021)) by STAR (ver.2.5.3a). The number of mapped reads was counted by featureCounts (1.5.2) and simultaneously normalized by the TMM method in the edgeR package in R [51]. The normalized expression levels processed to log2 and z-scoring were visualized using the pheatmap library. In the statistical analysis, Welch's t-test was performed between normalized gene expression levels of human samples and marmoset samples, and the resulting p values were processed with Bonferroni correction to obtain the adjusted p value. In the present study, adjusted p values less than 0.05 were defined as significant; adjusted p values processed to −log10 were visualized on the vertical axis by the ggplot2 library in R.

Western Blotting
Western blotting was performed using the Wes-Automated Western Blots with Simple Western (ProteinSimple) according to the manufacturer's introductions. As primary antibodies, we used polyclonal Rad51 H-92 antibody (1:50 dilution; sc-8349; Santa Cruz) and monoclonal α-tubulin antibody (1:25000 dilution; T9026; Sigma), which was used as an internal control for RAD51 protein quantification. ImageJ software was used to quantify the intensities of Rad51 (37 kDa) and α-tubulin (50 kDa) bands, and then RAD51 expression was normalized against α-tubulin expression.

Statistical Analysis
All data in this study are expressed as means ± S.D. Statistically significant differences were determined using Welch's t-test; p values < 0.05 are designated by *; p values <0.01 are designated by **, and are interpreted as statistically significant.

Comparative Transcriptomic Analysis of Human and Marmoset PSCs
Previously, we reported that cmESCs have an innately high HR activity [35]. In particular, extraordinarily high HR ratios for the 1st exon of PLP1 in a targeting experiment (92.3% with CRISPR-Cas9 against 88.6% without its use) were observed. We also observed high HR ratios using CRISPR-Cas9 in other loci, such as ACTB, PLP1 (targeting the 2nd, 5th, and 6th exons), FOXP2, PRDM1, DPPA3, and NANOS3 [20,35,36] (see Figure S1). Here, we investigated possible factors that might underlie this phenomenon.
Initially, we investigated HR-and NHEJ-related gene expression in human and marmoset PSCs. In hPSCs, several studies have shown that the HR ratio is less than 50%, generally around 30%, even with use of site-specific nucleases such as ZFN, TALEN, and CRISPR-Cas9 [31][32][33][34]. We used RNA-seq data of hESCs iPSCs deposited in databases [50] to compare gene expression with cmESCs as described previously [49]. We merged and normalized PSC RNA-seq data derived from both species.
HR-and NHEJ-related genes that can be used for comparing fold changes between hPSCs and cmESCs have been listed by hsa03440 and ko03450 in KEGG (https://www. genome.jp/kegg/pathway.html (accessed on 1 December 2021)). Some related genes are absent from the gene lists due to the incomplete gene assembly of the latest version of the marmoset genome (Callithrix_jacchus_cj1700_1.1). Here, we analyzed 37 HR-related and 12 NHEJ-related genes ( Figure 1A-D, Figures S2 and S3). As summarized in Supplementary Data S1, eleven genes (RAD51D, BRCA1, BRCA2, BABAM1, RAD51, RAD51C, POLD2, RAD51B, MUS81, POLD1, and XRCC3) were significantly up-regulated in cmESCs ( Figure 1A), whereas eleven others (RPA2, ATM, XRCC2, SSBP1, RPA3, PALB2, BRIP1, NBN, RAD54B, UMC1, and BRCC3) were down-regulated ( Figure 1B). With regard to NHEJ-related genes, six genes (XRCC6, PRKDC, POLM, XRCC5, RAD50, and DNTT) were significantly up-regulated in cmESCs ( Figure 1C), and five others (FEN1, DOLRE1C, NHEJ1, XRCC4, and POLL) were down-regulated ( Figure 1D). In light of the high HR activity in cmESCs, we then focused on HR-related genes that showed increased expression in cmESCs compared to hPSCs. To validate the results of the transcriptome analysis, we performed an interspecies qPCR analysis using primer sets specifically designed for these human and marmoset genes. We also designed primers specific for human and marmoset GAPDH for normalization. Preliminary screens using the designed primers showed that an interspecies comparison of expressions was not feasible for several genes (BABAM1 and POLD1/2) owing to a lack of accuracy based on the post-qPCR melt curve analysis (data not shown).

Enhancement of the HR and RI Ratio with CRISPR-Cas9 in hiPSCs by Overexpression of the Defined Factors
To explore the effect of high expression of the five HR-related genes (RAD51D, RAD51C, BRCA2, RAD51, and BRCA1) in cmESCs, we induced overexpression of the genes in a gene targeting experiment with HPRT in the male hiPSC line etKA4 [44]. The HPRT protein catalyzes the salvage pathway, synthesizing inosine monophosphate and guanosine monophosphate from hypoxanthine and guanine, respectively [52]. HPRT deficiency results in the loss of susceptibility for 6TG, a toxic analog of guanine [53,54]. We selected the HPRT targeting system as it has been frequently used to assess the HR ratio in mammalian male PSCs [24,55,56].  We constructed a knock-in/knock-out system for the human HPRT gene, which is located on the X chromosome ( Figure 3A,B). The targeting vector (HPRT-TV) harbored a neomycin-resistance (PGK-Neo) cassette flanked by 3.0 kb 5' homology and 12.8 kb 3' homology arms ( Figure 3A). Following KI, the 2nd exon of HPRT was completely replaced with a PGK-Neo cassette, which resulted in the loss of functional mRNA expression from the HPRT Neo allele ( Figure 3A). Initial G418 selection (both homologous recombinants and non-recombinants survived) and subsequent 6TG selection (only homologous recombinants survived) enabled robust quantification of the HR ratio without the necessity of genotyping individual clones ( Figure 3B). In addition, we constructed a PX459 (Addgene #62988)-based Cas9/gRNA vector [39] containing the sgRNA sequence for the 2nd intron of HPRT, which did not recognize HPRT-TV.

Enhancement of the HR and RI Ratio with CRISPR-Cas9 in hiPSCs by Overexpression of the Defined Factors
To explore the effect of high expression of the five HR-related genes (RAD51D, RAD51C, BRCA2, RAD51, and BRCA1) in cmESCs, we induced overexpression of the genes in a gene targeting experiment with HPRT in the male hiPSC line etKA4 [44]. The HPRT protein catalyzes the salvage pathway, synthesizing inosine monophosphate and guanosine monophosphate from hypoxanthine and guanine, respectively [52]. HPRT deficiency results in the loss of susceptibility for 6TG, a toxic analog of guanine [53,54]. We selected the HPRT targeting system as it has been frequently used to assess the HR ratio in mammalian male PSCs [24,55,56].
We constructed a knock-in/knock-out system for the human HPRT gene, which is located on the X chromosome ( Figure 3A,B). The targeting vector (HPRT-TV) harbored a neomycin-resistance (PGK-Neo) cassette flanked by 3.0 kb 5' homology and 12.8 kb 3' homology arms ( Figure 3A). Following KI, the 2nd exon of HPRT was completely replaced with a PGK-Neo cassette, which resulted in the loss of functional mRNA expression from the HPRT Neo allele ( Figure 3A). Initial G418 selection (both homologous recombinants and non-recombinants survived) and subsequent 6TG selection (only homologous recombinants survived) enabled robust quantification of the HR ratio without the necessity of genotyping individual clones ( Figure 3B). In addition, we constructed a PX459 (Addgene #62988)-based Cas9/gRNA vector [39] containing the sgRNA sequence for the 2nd intron of HPRT, which did not recognize HPRT-TV. As it is possible that the genomic cleavage of the HPRT 2nd intron after transfection of the Cas9/gRNA vector and subsequent NHEJ or MMEJ-mediated introduction of small and large deletion could produce an undesired knock-out allele, we initially tested transfection of only the Cas9/gRNA vector into the etKA4 hiPSCs. In this initial test, no 6TG-resistant colonies were obtained from 1 × 10 7 transfected cells (n = 3), showing that the NHEJ or MMEJ-mediated deletion in the intronic region has a negligible effect with regard to the assessment of the HR ratio in the hiPSCs.
After co-transfection of the Cas9/gRNA vector and HPRT-TV, and serial G418 and 6TG selection, we confirmed that all analyzed G418 and 6TG-resistant (NeoR+6TGR) clones were hemizygous HPRT Neo recombinants by Southern blotting ( Figure 3C).
Last, we performed additional KI experiments in another locus, PROX1. We used the Cas9/gRNA vector and PROX1-Venus targeting vector (PROX1-TV), which was slightly modified from a previous study [20]. By the construct, 2A-Venus and a puromycin-resistance cassette are introduced into the termination codon (exon 5) of the PROX1 gene ( Figure S5A). We transfected the Cas9/gRNA and PROX1-TV with or without the overexpression vectors of five (RAD51, RAD51C, RAD51D, BRCA1, and BRCA2) factors. Following puromycin selection, hiPSC colonies, putative KI, or WT clones were genotyped by PCR ( Figure S5B). The results of the experiments are summarized in Figure S5C-3/12 for control (no overexpression), 5/12 for RAD51 overexpression, 6/12 for RAD51C overexpression, 7/12 for RAD51D or BRCA2 overexpression, 8/12 for BRCA1, or four factor (RAD51C/D and BRCA1/2) overexpression. Again, the overexpression of four factors enhanced KI efficiency in hiPSCs, which shows the robust applicability of the technology for further applications. The precise integration of 2A-Venus was confirmed by Sanger Sequencing ( Figure S5D). The precise knock-in at the HPRT locus (described in Figure 3) was also confirmed by genotyping PCR and Sanger sequencing ( Figures S6 and S7). We also note that the overexpression of BRCA2 may have directly or indirectly affected the perturbation of hiPSCs' cell cycle ( Figure S8).

Discussion
In this study, through comparative analyses of gene expression in human and marmoset PSCs, we have identified four genes (RAD51C, RAD51D, BRCA1, and BRCA2) whose single and multiple overexpression increased HR ratios in hiPSCs. Intriguingly, we also observed that overexpression of RAD51 did not enhance the HR ratio in hiPSCs; an alternative explanation is that RAD51 overexpression may cancel the effect of the enhancement of the HR ratio by the other four genes. In the Supplementary Discussion, we further discuss how these genes involve in the HR machinery, and the discrepancy of the effect of RAD51 overexpression from the part of previous studies. Results presented here also suggest the possibility of a vice versa effect. In fact, we demonstrated the overexpression of four factors (RAD51C, RAD51D, BRCA1, and BRCA2), which were highly expressed in cmESCs, and which contributed to the enhancement of HR ratios in hPSCs. Thus, it is also possible that overexpression of several NHEJ factors, which were lowly expressed in cmESCs (including FEN1, DCLRE1C (Artemis), NHEJ1, and XRCC4), may contribute to decreased HR ratios in hPSCs. Further analyses are required to evaluate the robust effects of the four HR factors, such as effects on KI in other loci, and in different cell lines and species; nevertheless, our investigation has demonstrated that overexpression of these factors may ameliorate the HR ratio with CRISPR-Cas9 in hiPSCs. We also note that, in the present study, we could not scrutinize the risk of HR factor overexpression for the usage of iPSCs in other experimental settings, including transplantation and drug-screening analysis.
In clinical settings, although knock-in technology in donor PSCs is beneficial due to the highly customizability, constitutive overexpression of exogenous gene(s) may impose potential risks, including tumorigenicity and genome instability. In this context, the critical time window of HR-factor overexpression for increasing HR ratios should be assessed in further studies.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11030360/s1, Figure S1: Gene targeting in the marmoset NANOS3 gene locus; Figure S2: Normalized genes expression of HR-related genes in human and marmoset PSCs; Figure S3: Normalized genes expression of NHEJ-related genes in human and marmoset PSCs; Figure S4: Western blotting analysis of RAD51 protein in human and marmoset PSCs; Figure S5: Gene targeting in the human PROX1 gene locus; Figure S6: Genotyping PCR analysis of WT and G418/6-TG-resistant iPSCs in the HPRT locus; Figure S7: DNA sequencing analysis of the PCR-amplified HPRT Neo allele in G418 and 6-TG resistant iPSCs; Figure   Institutional Review Board Statement: All animal experiments were performed in accordance with the guidelines for laboratory animals set forth by the National Institutes of Health, and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and were approved by the Institutional Animal Care and Use Committee of RIKEN Institute under the approval No. H27-2-306(4).

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.