Reduction of cellulose crystallization by CRISPR/Cas9 -mediated mutagenesis in the P-CR domain of CESA4 subunit of white poplar (Populus alba L.)

Abstract Cellulose is the most abundant linear homopolysaccharide in nature, which has always raised great attention from investigators and industrial investors for use in wood and paper pulp industries, biofuel, and nanocellulose products due to its semi-crystalline nature and unique physicochemical properties. However, the cell wall recalcitrance and high cellulose crystallinity of wood biomass are the most challenging issues during cellulose extraction and its saccharification in bioethanol and nanocellulose production industries. Here, we have investigated the effect of CRISPR/Cas9-mediated mutagenesis on the Pro435 and Try436 residues at the binding site of PalCESA4-specific P-CR domain from white poplar. As the result, we generated a PalCESA4P435del_W436del homozygous T0 mutant plant with normal growth that showed a significant decrease in cell wall area (21.89%), cell wall thickness (7.5%), cellulose content (~ 44%), and cellulose crystallinity (19.5%) compared to the WT plant. Our findings reveal a promising approach to achieving genetically edited woods with suitable physicochemical properties of cellulose microfibrils in different plant species for industrial applications. IntroductionCellulose is the most abundant linear homopolymer in wood biomass of woody plants. Due to its semi-crystalline nature, it uses in various industrial applications, including wood and paper, textile, pharmaceutical and medicinal, agriculture, biofuel, and bio-nanomaterials (Littlewood et al., 2014; Trache et al., 2020). Despite its wide applications, high cellulose crystalization is the most important challenge in the production processes of bioethanol and nanocellulose from wood biomass (Vermerris and Abril, 2015). As the previous reports associated with the cellulose biosynthesis pathway in Arabidopsis and poplar, the CrI and DP of cellulose, the number of microfibrils in the cell wall architecture is highly influenced by the arrangement of cellulose synthase A (CESA) subunits during the formation of the cellulose synthase complex (CSC) (Luan et al., 2011; Scanlon and Timmermans, 2013; Speicher et al., 2018). In our previous study, we identified the strong binding site containing four amino acids Pro435, Trp436, Pro437, and Gly438 in the P-CR domain of poplar CESA4 subunit, which is involved in the CESA4 and CESA8 heterodimerization. The CRISPR/Cas9-mediated mutagenesis in Trp436 and Pro437 residues could alter plant growth and photosynthesis rates, a cell wall thickness of xylem and phloem cells, lignocellulose content, CrI, and DP of cellulose fibers in poplar (Nayeri et al., 2022). Materials and Methods The CESA4-specific sgRNA (5′-CAGGATGGTACCCCCATGGCCTGG-3′) was designed using CRISPRdirect, CHOPCHOP, and CRISPOR-Tefor tools. The genome sequence data of P. trichocarpa v 4.1 was used to determine the sgRNA on-target and off-target sites. The molecular cloning of the recombinant CRISPR/Cas9 expression vector (Figure 1) was performed according to the method described by Nayeri et al., 2022. The pulvini tTCL explants from the petioles of 4th nodes close to apical buds of P. alba L. were transformed using competent bacterial cells of Agrobacterium tumefaciens strain LBA4404 as described by Nayeri et al., 2022. The T0 plants were screened and validated using PCR analysis of bar and pcoCas9 genes. The bacterial contaminations of to plants were examined using PCR analysis of the Vir G gene. The screening of T0 mutants and identification of mutation types in the PalCESA4-sgRNA target site gene were performed using heteroduplex PCR genotyping methods (Guo et al., 2018), amplification of PCR fragments using mutation site-specific primers (MSBSP-PCR) (Bhattacharya and Van Meir, 2019) and sanger sequencing analysis. The sequence data of primer sets were presented in Table 1. The wood anatomy of the T0 mutant was investigated using light microscopy (LM) and field emission scanning electron microscopy (FE-SEM) analysis. The contents of lignocellulosic components were determined according to the TAPPI international standard guidelines. The cellulose crystallinity index (CrI) and its degree of polymerization (DPN, DPW, PDI) were determined using XRD analysis patterns and gel permeation chromatography (GPC), respectively. The statistical analysis was performed using one-way ANOVA analysis of variance and mean comparison based on LSD posthoc's minimum significant difference using SPSS IBM v. 22.0 (SPSS Inc, Chicago, USA). The maximum significance level was set at 5% (p-value ≤0.05). Results and discussionAs shown in the PCR analysis results, 14 out of 15 regenerated T0 plantlets had DNA bands of PCR products belonging to bar and pcoCas9 genes. Furthermore, 11 out of 14 T0 plants showed a lack of DNA bands from the PCR2 products (196 bp), which indicates homozygous or biallelic mutations in T0 mutants. In the rest of the T0 plants, no mutation or heterozygous mutations were observed due to the sharp DNA bands from the PCR2 products. The heteroduplex genotyping results showed the double DNA band of CESA-PCR3 fragments (160 bp) in 5% agarose gel, indicating heterozygous mutation in the T0 mutant poplar. However, the DNA hybridization in potent biallelic/homozygous T0 mutants indicates homozygous mutation in all 11 T0 mutants. Among the T0 mutants, we identified a homozygous T0 mutant plant (L7) with double deletion mutations in P435 and W436 residues (Figure 3E). As the wood anatomy analysis results, the Vd value of the homozygous palCESA4P435del_W436del (L7) T0 plant showed a significant decrease of 74.85% compared with the WT. The cell wall area (21.89%), cell wall thickness (7.5%), cellulose content (approximately 44%), and CrI of cellulose (19.5%) significantly decreased. According to the previous reports, the knockdown or knockout of PtriCESA4, PtriCESA7a/b, and PtriCESA8a/b genes cause a remarkable decrease in the thickness of the cell wall and cellulose content (~90%), resulting in the production of unhealthy and abnormal mutants (Abbas et al., 2020; Xu et al., 2021). Our previous findings reveal that substitution and deletion of Trp436 and Pro437 residues in the binding site of CESA4-specific P-CR domain led to a 13-25% decrease in the cellulose CrI from wood biomass of T0 poplar mutant (Nayeri et al., 2022). ConclusionThese findings can be used in the production of gene-edited plants using the CRISPR/Cas9 system with desirable traits such as low cellulose crystalization, leading to saving in costs and time in the pure cellulose extraction process and preparation of cellulose-based biomaterials. Therefore, this isa promising technology for the mass production of cellulose-based products, which increases export volume and develop new and environmental-friendly technologies in the country. AcknowledgementThis article was extracted from the final report of the research project under contract number 346/27/D, which was applied to the research grant of the University of Tabriz, Tabriz, Iran. Our special thanks to Iranian Research Institute for Information Science and Technology (IranDoc) for technical and financial suports of our genetic engineering lab, the center of graduate education, faculty of agriculture, university of Tabriz, Tabriz-51666, Iran IR.