Highly efficient CRISPR systems for loss-of-function and gain-of-function research in pear calli

Abstract

CRISPR/Cas systems have been widely used for genome engineering in many plant species. However, their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only a few reports on the application of the CRISPR/Cas9 system in pear have been documented, and have shown very low editing efficiency. Here we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand the targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taking these results together, we have built a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.

References

[1] Hsu PD, Lander ES, Zhang F . Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014; 157: 1262-78.

[2] Hille F, Richter H, Wong SP et al. The biology of CRISPR-Cas: backward and forward. Cell. 2018; 172: 1239-59.

[3] Zhang Y, Malzahn AA, Sretenovic S et al. The emerging and uncultivated potential of CRISPR technology in plant science. Nat Plants. 2019; 5: 778-94.

[4] Zhou J, Li D, Wang G et al. Application and future perspective of CRISPR/Cas9 genome editing in fruit crops. J Integr Plant Biol. 2020; 62: 269-86.

[5] Zhu H, Li C, Gao C . Applications of CRISPR-Cas in agriculture and plant biotechnology. Nat Rev Mol Cell Biol. 2020; 21: 661-77.

[6] Chen K, Wang Y, Zhang R et al. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol. 2019; 70: 667-97.

[7] Wang T, Zhang H, Zhu H . CRISPR technology is revolutionizing the improvement of tomato and other fruit crops. Hortic Res. 2019; 6: 77.

[8] Li T, Yang X, Yu Y et al. Domestication of wild tomato is accelerated by genome editing. Nat Biotechnol. 2018; 36: 1160-3.

[9] Varkonyi-Gasic E, Wang T, Voogd C et al. Mutagenesis of kiwifruit CENTRORADIALIS-like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering. Plant Biotechnol J. 2019; 17: 869-80.

[10] Xing S, Chen K, Zhu H et al. Fine-tuning sugar content in strawberry. Genome Biol. 2020; 21: 230.

[11] Zhang J, Zhang H, Li S et al. Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9. J Integr Plant Biol. 2021; 63: 1649- 63.

[12] Wang L, Chen S, Peng A et al. CRISPR/Cas9-mediated editing of CsWRKY22 reduces susceptibility to Xanthomonas citri subsp. citri in Wanjincheng orange (Citrus sinensis (L.) Osbeck) . Plant Biotechnol Rep. 2019; 13: 501-10.

[13] Tao H, Shi X, He F et al. Engineering broad-spectrum disease-resistant rice by editing multiple susceptibility genes. J Integr Plant Biol. 2021; 63: 1639-48.

[14] Deng L, Wang H, Sun C et al. Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. J Genet Genomics. 2018; 45: 51-4.

[15] Yan S, Chen N, Huang Z et al. Anthocyanin fruit encodes an R2R3-MYB transcription factor, SlAN2-like, activating the transcription of SlMYBATV to fine-tune anthocyanin content in tomato fruit. New Phytol. 2020; 225: 2048-63.

[16] Ye M, Peng Z, Tang D et al. Generation of self-compatible diploid potato by knockout of S-RNase. Nat Plants. 2018; 4: 651-4.

[17] Ma X, Zhang Q, Zhu Q et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015; 8: 1274-84.

[18] Wang ZP, Xing HL, Dong L et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 2015; 16: 144.

[19] Yan L, Wei S, Wu Y et al. High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9 system. Mol Plant. 2015; 8: 1820-3.

[20] Li G, Sretenovic S, Eisenstein E et al. Highly efficient C-to-T and A-to-G base editing in a Populus hybrid. Plant Biotechnol J. 2021; 19: 1086-8.

[21] Ren C, Liu Y, Guo Y et al. Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters. Hortic Res. 2021; 8: 52.

[22] Jinek M, Chylinski K, Fonfara I et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337: 816-21.

[23] Zetsche B, Gootenberg JS, Abudayyeh OO et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163: 759-71.

[24] Ming M, Ren Q, Pan C et al. CRISPR-Cas12b enables efficient plant genome engineering. Nat Plants. 2020; 6: 202-8.

[25] Tang X, Lowder LG, Zhang T et al. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants. 2017; 3: 17018.

[26] Zhong Z, Zhang Y, You Q et al. Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites. Mol Plant. 2018; 11: 999-1002.

[27] Wang Q, Alariqi M, Wang F et al. The application of a heat-inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G. hirsutum) plants . Plant Biotechnol J. 2020; 18: 2436-43.

[28] Zhang Y, Ren Q, Tang X et al. Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems. Nat Commun. 2021; 12: 1944.

[29] Konermann S, Brigham MD, Trevino AE et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2015; 517: 583-8.

[30] Lowder LG, Zhang D, Baltes NJ et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015; 169: 971-85.

[31] Chavez A, Tuttle M, Pruitt BW et al. Comparison of Cas9 activators in multiple species. Nat Methods. 2016; 13: 563-7.

[32] Li Z, Zhang D, Xiong X et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nat Plants. 2017; 3: 930-6.

[33] Liao HK, Hatanaka F, Araoka T et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell. 2017; 171: 1495-1507.e15.

[34] Lowder LG, Zhou J, Zhang Y et al. Robust transcriptional activation in plants using multiplexed CRISPR-Act2.0 and mTALE-act systems. Mol Plant. 2018; 11: 245-56.

[35] Pan C, Wu X, Markel K et al. CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants. Nat Plants. 2021; 7: 942-53.

[36] Wu J, Wang Z, Shi Z et al. The genome of the pear (Pyrus bretschneideri Rehd.) . Genome Res. 2013; 23: 396-408.

[37] Charrier A, Vergne E, Dousset N et al. Efficient targeted mutagenesis in apple and first time edition of pear using the CRISPR-Cas9 system. Front Plant Sci. 2019; 10: 40.

[38] Cheng J, Zhang M, Tan B et al. A single nucleotide mutation in GID1c disrupts its interaction with DELLA1 and causes a GA-insensitive dwarf phenotype in peach. Plant Biotechnol J. 2019; 17: 1723-35.

[39] Yao G, Ming M, Allan AC et al. Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. Plant J. 2017; 92: 437-51.

[40] Lu S, Zhang Y, Zhu K et al. The citrus transcription factor CsMADS6 modulates carotenoid metabolism by directly regulating carotenogenic genes. Plant Physiol. 2018; 176: 2657-76.

[41] An JP, Yao JF, Xu RR et al. Apple bZIP transcription factor MdbZIP44 regulates abscisic acid-promoted anthocyanin accumulation. Plant Cell Environ. 2018; 41: 2678-92.

[42] Bai S, Tao R, Tang Y et al. BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol J. 2019; 17: 1985-97.

[43] Bai S, Tao R, Yin L et al. Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J. 2019; 100: 1208-23.

[44] Xue C, Yao JL, Xue YS et al. PbrMYB169 positively regulates lignification of stone cells in pear fruit. J Exp Bot. 2019; 70: 1801-14.

[45] Wang R, Xue Y, Fan J et al. A systems genetics approach reveals PbrNSC as a regulator of lignin and cellulose biosynthesis in stone cells of pear fruit. Genome Biol. 2021; 22: 313.

[46] Tao S, Khanizadeh S, Zhang H et al. Anatomy, ultrastructure and lignin distribution of stone cells in two Pyrus species. Plant Sci. 2009; 176: 413-9.

[47] Papikian A, Liu W, Gallego-Bartolome J et al. Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems. Nat Commun. 2019; 10: 729.

[48] Liu H, Su J, Zhu Y et al. The involvement of PybZIPa in light-induced anthocyanin accumulation via the activation of PyUFGT through binding to tandem G-boxes in its promoter. Hortic Res. 2019; 6: 134.

[49] Bae S, Park J, Kim JS . Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014; 30: 1473-5.

[50] Nishitani C, Hirai N, Komori S et al. Efficient genome editing in apple using a CRISPR/Cas9 system. Sci Rep. 2016; 6: 31481.

[51] Zhou J, Wang G, Liu Z . Efficient genome editing of wild strawberry genes, vector development and validation. Plant Biotechnol J. 2018; 16: 1868-77.

[52] Xing H-L, Dong L, Wang ZP et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014; 14: 327.

[53] Ren Q, Sretenovic S, Liu G et al. Improved plant cytosine base editors with high editing activity, purity, and specificity. Plant Biotechnol J. 2021; 19: 2052-68.

[54] Lee K, Zhang Y, Kleinstiver BP et al. Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize. Plant Biotechnol J. 2019; 17: 362-72.

[55] Li J, Zhang S, Zhang R et al. Efficient multiplex genome editing by CRISPR/Cas9 in common wheat. Plant Biotechnol J. 2021; 19: 427-9.

[56] Randall LB, Sretenovic S, Wu Y et al. Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor. Plant Physiol. 2021; 187: 73-87.

[57] Jia H, Wang N . Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One. 2014; 9: e93806.

[58] Nakajima I, Ban Y, Azuma A et al. CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS One. 2017; 12: e0177966.

[59] Wang Z, Wang S, Li D et al. Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit. Plant Biotechnol J. 2018; 16: 1424-33.

[60] Kaur N, Alok A, Shivani et al. CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome. Funct Integr Genomics. 2018; 18: 89-99.

[61] Naim F, Dugdale B, Kleidon J et al. Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9. Transgenic Res. 2018; 27: 451-60.

[62] Xing S, Jia M, Wei L et al. CRISPR/Cas9-introduced single and multiple mutagenesis in strawberry. J Genet Genomics. 2018; 45: 685-7.

[63] Wilson FM, Harrison K, Armitage AD et al. CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry. Plant Methods. 2019; 15: 45.

[64] Wang X, Tu M, Wang D et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant Biotechnol J. 2018; 16: 844-55.

[65] Martin-Pizarro C, Trivino JC, Pose D . Functional analysis of the TM6 MADS-box gene in the octoploid strawberry by CRISPR/Cas9-directed mutagenesis. J Exp Bot. 2019; 70: 885-95.

[66] Peng A, Chen S, Lei T et al. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnol J. 2017; 15: 1509-19.

[67] Wang M, Mao Y, Lu Y et al. Multiplex gene editing in rice using the CRISPR-Cpf1 system. Mol Plant. 2017; 10: 1011-3.

[68] Li B, Rui H, Li Y et al. Robust CRISPR/Cpf1 (Cas12a)-mediated genome editing in allotetraploid cotton (Gossypium hirsutum) . Plant Biotechnol J. 2019; 17: 1862-4.

[69] Li B, Liang S, Alariqi M et al. The application of temperature sensitivity CRISPR/LbCpf1 (LbCas12a) mediated genome editing in allotetraploid cotton (G. hirsutum) and creation of nontransgenic, gossypol-free cotton . Plant Biotechnol J. 2021; 19: 221-3.

[70] Jia H, Orbovic V, Wang N . CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnol J. 2019; 17: 1928-37.

[71] Malzahn AA, Tang X, Lee K et al. Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis . BMC Biol. 2019; 17: 9.

[72] Teng F, Cui T, Feng G et al. Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov. 2018; 4: 63.

[73] Xu J, Hua K, Lang Z . Genome editing for horticultural crop improvement. Hortic Res. 2019; 6: 113.

[74] Meng X, Yu H, Zhang Y et al. Construction of a genome-wide mutant library in rice using CRISPR/Cas9. Mol Plant. 2017; 10: 1238-41.

[75] Lu Y, Ye X, Guo R et al. Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system. Mol Plant. 2017; 10: 1242-5.

[76] Jacobs TB, Zhang N, Patel D et al. Generation of a collection of mutant tomato lines using pooled CRISPR libraries. Plant Physiol. 2017; 174: 2023-37.

[77] Liu HJ, Jian L, Xu J et al. High-throughput CRISPR/Cas9 mutagenesis streamlines trait gene identification in maize. Plant Cell. 2020; 32: 1397-413.

[78] Bai M, Yuan J, Kuang H et al. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean. Plant Biotechnol J. 2020; 18: 721-31.

[79] An JP, Qu FJ, Yao JF et al. The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hortic Res. 2017; 4: 17023.

[80] An JP, An XH, Yao JF et al. BTB protein MdBT2 inhibits anthocyanin and proanthocyanidin biosynthesis by triggering MdMYB9 degradation in apple. Tree Physiol. 2018; 38: 1578-87.

[81] You Q, Zhong Z, Ren Q et al. CRISPRMatch: an automatic calculation and visualization tool for high-throughput CRISPR genome-editing data analysis. Int J Biol Sci. 2018; 14: 858-62.

[82] Yamagishi Y, Yoshimoto J, Uchiyama H et al. In vitro induction of secondary xylem-like tracheary elements in calli of hybrid poplar (Populus sieboldii × P. grandidentata) . Planta. 2013; 237: 1179-85.