Establishment of high‐efficiency genome editing in white birch (<i>Betula platyphylla</i> Suk.)

Abstract

To cope with increasing climate change, growing demands of society, the long breeding cycles and various stresses, that is necessary to establish molecular breeding system, through transgene and genome editing technology, for natural forest tree species. White birch (Betula platyphylla Suk.), belonging to the Betulaceae family, is distributed in the Northeast China forest with ecological and economic significance (Chen et al., 2021). In contrast, there have been limited studies on the biology of white birch and the challenge of lower transgene and editing efficiency prompted us to further concentrate on optimizing the genome editing system in this species. Since the efficiency of genome editing can vary depending on the genotypes of the receptor materials, which is mainly determined by regeneration rates of tissue culture, we conducted a screening process based on the superior white birch strain DL-1 that has higher differentiation efficiency, growth speed and biomass than other 19 lines according to our previous analysis (Figure 1a,b; Tu et al., 2022). We specifically investigate the activity of Pol III promoters to drive sgRNA targeting the phytoene desaturase (PDS) gene by DL-1 matured zygotic embryo infection (Figure S1). For this purpose, we constructed binary vectors in pSCI using the AtU6-26 promoter from Arabidopsis (Mao et al., 2013), the GmU6-5 from soybean (Wang, Yiting (Wang, Y.T.), Zhang, Qingzhu (Zhang, Q.Z.), Zheng, Zhimin (Zheng, Z.M.) and 16 endogenous Pol III promoters from white birch, including eight U6 promoters, 7SL and seven U3, to drive sgRNA expression (Figure S2–S5 and Table S1). After obtaining the basta-resistant seedlings, we proceeded to extract genomic DNA from transgenic lines that contained binary vectors (Pol III + sgRNA + Cas9) as well as empty pSC1 vector (containing only Cas9), then identified the positive lines by PCR with M13R and sgRNA primers, and then employed PCR amplification of target sequence to confirm whether mutation had occurred in any of the transgenic lines with binary vectors (Pol III + sgRNA + Cas9). The sgRNA target site located in the second exon (sgRNA1) or the fourth exon(sgRNA2) of PDS gene was screened in transgenic lines using Sanger sequencing and then screened the high-efficiency promoters. We observed a total of 670 editing events, and it was evident that the genome editing efficiency varied significantly with various Pol III promoters in the first generation (T0) (Figure 1c). For the exogenous Pol III promoters, AtU6-26 (55.56%, n = 45) is higher than GmU6-5 (31.82%, n = 44, P < 0.05, Chi-square test) in the sgRNA2 target site. Except U3-6, U3-7, U6-3 and U6-4, other endogenous Pol III promoters consistently exhibited editing efficiencies approximately ~1–2 times (55.81%–92.00%, n = 43–53) higher than that of AtU6-26 (P < 0.05, Chi-square test, Figure 1c; Table S2). For the endogenous Pol III promoters, we obtained a total of 574 editing events. The editing outcomes revealed that the main types of mutations were short deletions ranging from 1 to 10 nts (76.82%, 232/302) when utilizing BpU6 promoters to drive sgRNA2 activity. Moreover, deletions from approximately 10 to 100 nts (14.24%, 43/302) were observed using U6 promoters (Figure 1d; Figure S7a). The next most frequent mutation types were nucleotide base insertions (7.28%, 22/302, Figure S7a). The percentage of base replacement mutations is 1.66% (5/302, Figure 1c), which is a common occurrence in non-homologous end joining (NHEJ) DNA repair (Manghwar et al., 2019). When using BpU3 promoters to drive sgRNA1 expression, the number of 1 to 10 nts small deletion is 176 (82.63%, 176/213), except for U3-2 promoter, which did not produce edited lines (Figure 1c,d; Figure S7b and Table S2). A total of 15 (7.04%, 15/213) deletions from approximately 10 to 100 nts were observed in U3-1, U3-3, U3-4 and U3-5 promoters (Figure 1d; Figure S7b). Notably, U3-3-mediated gene editing events included large fragment deletions, such as a 358-nt, or a 240-nt or a 117-nt deletion in U3-transgenic lines (Figure 1e; Figure S7b). A similar pattern was observed when using the Bp7SL promoter to drive sgRNA2 expression (n = 59). Furthermore, we observed that the frequencies of non-heterozygous mutations were higher when using endogenous promoters compared to AtU6-26 or GmU6-5 for sgRNA2 expression in white birch. Particularly, the U3-3 promoter resulted in over 2-fold increases (2.04- and 2.29-fold, respectively) in non-heterozygous mutation frequencies (P < 0.05, Chi-square test, Figure 1c; Table S2). Based on the biallelic and homozygous mutation frequency results, we classified mutations into three types, m (missense), n (nonsense) and s (synonymous), based on their putative protein mutations (Figure 1c; Figure S8). We observed that transgenic plants carrying ‘m/n’ and ‘n/n’ genotypes were the most prevalent among edited lines (Figure 1c). Notably, transgenic lines carrying endogenous Pol III promoters U3-7, U3-3 or U3-1 exhibited approximately ~1–2 times (90.01%–100.00%, n = 1–18) higher proportions of ‘m/n’ and ‘n/n’ lines compared to transgenic lines carrying AtU6-26 or GmU6-5 promoters (P < 0.05, Chi-square test). Additionally, we observed that the frequencies of plants carrying only the ‘n/n’ genotype in transgenic lines carrying U3-3, U3-1, U6-1, U3-4 or U3-5 promoters were approximately ~1–2 times higher than those in the transgenic line carrying the AtU6-26 promoter (P < 0.05, Chi-square test, Figure 1c). The analysis results indicated that genotypes containing the ‘n’ allele play a crucial role in albino phenotypic variation (Figure 1f,g). Transgenic lines displayed variegated phenotypes when the ‘n’ genotype was heterozygous, while they exhibited an albino phenotype in ‘n/n’ homozygotes. In contrast, the ‘m’ allele did not contribute significantly to variations in the albino phenotype, as transgenic plants appeared pale green regardless of whether they carried homozygous ‘m/m’ or heterozygous ‘m’ genotypes (Figure 1f,g). We identified sgRNA2 in exon 4, within the conserved domains of PDS (Figure 1f), further indicating that disruption of these conserved domains greatly impacts gene function. Figure 1g illustrates how the phenotypes of 11 transgenic lines correspond to the variant genotypes and protein sequences shown in Figure 1f. Based on our analysis, we recommend utilizing the BpU6-6 promoter for future genome editing of white birch, which has higher editing efficiency. In summary, the present study successfully used high-efficiency endogenous promoters to drive sgRNA expression of target loci in white birch. To our knowledge, there has been no prior research on screening for efficient Pol III promoters from natural forest trees, specifically white birch. Furthermore, the identification of efficient promoters could greatly aid in the establishment of a multiple-gene editing system to target multiple genes. Our findings provide valuable and practical support for the development of a multiple-gene editing system and molecular breeding in white birch as well as other forest trees. The present study was supported by the National Key R&D Program of China during the 14th Five-year Plan Period (2021YFD2200102). The authors declare that they have no competing interests. Prof. Zhimin Zheng designed the experimental details, conducted the experiments and wrote the manuscripts. Dawei Cheng and Yueying Liu performed the experiments, analysed the data, designed the figures and wrote the manuscript. Yi Wang, Lesheng Cao and Siyao Wu participated in some of the bioinformatics analysis, vector construction and genetic transformation experiments. Prof. Qingzhu Zhang, Prof. Li-nan Xie and Song Yu contributed to some of the data analysis and discussion in this article. Prof. Guifeng Liu, Prof. Jing Jiang and Huiyu Li provided the research materials and guided the genetic transformation experiments. Figure S1–S8 Supplementary Figures. Table S1–S2 Supplementary Tables. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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