Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9

Abstract

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3^′ end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.

References

[1] Li S, Li J, He Y et al. Precise gene replacement in rice by RNA transcript-templated homologous recombination. Nat Biotechnol. 2019; 37: 445-50.

[2] Cermak T, Baltes NJ, Cegan R et al. High-frequency, precise modification of the tomato genome. Genome Biol. 2015; 16: 232.

[3] Bewg WP, Ci D, Tsai C-J . Genome editing in trees. From multiple repair pathways to long-term stability. Front Plant Sci. 2018; 9: 1732.

[4] Zhou X, Jacobs TB, Xue L-J et al. Exploiting SNPs for biallelic CRISPR mutations in the outcrossing woody perennial Populus reveals 4-coumarate:CoA ligase specificity and redundancy. New Phytol. 2015; 208: 298-301.

[5] Schiml S, Fauser F, Puchta H . The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 2014; 80: 1139-50.

[6] Li J, Meng X, Zong Y et al. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nat Plants. 2016; 2: 16139.

[7] Yang D, Scavuzzo MA, Chmielowiec J et al. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep. 2016; 6: 21264.

[8] Gil-Humanes J, Wang Y, Liang Z et al. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J. 2017; 89: 1251-62.

[9] Svitashev S, Schwartz C, Lenderts B et al. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun. 2016; 7: 13274.

[10] van Vu T, Sivankalyani V, Kim EJ et al. Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato. Plant Biotechnol J. 2020; 18: 2133-43.

[11] Movahedi A, Zhang J, Gao P et al. Expression of the chickpea CarNAC3 gene enhances salinity and drought tolerance in transgenic poplars. Plant Cell Tissue Organ Cult. 2015; 120: 141-54.

[12] Movahedi A, Zhang JX, Amirian R et al. An efficient Agrobacterium-mediated transformation system for poplar. Int J Mol Sci. 2014; 15: 10780-93.

[13] An Y, Geng Y, Yao J et al. Efficient genome editing in Populus using CRISPR/Cas12a. Front Plant Sci. 2020; 11: 593938.

[14] Ali Z, Shami A, Sedeek K et al. Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice. Commun Biol. 2020; 3: 44.

[15] Tran NT, Bashir S, Li X et al. Enhancement of precise gene editing by the association of Cas9 with homologous recombination factors. Front Genet. 2019; 10.

[16] Friesner J, Britt AB . Ku80- and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration. Plant J. 2003; 34: 427-40.

[17] Maruyama T, Dougan SK, Truttmann MC et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015; 33: 538-42.

[18] Pierce AJ, Hu P, Han M et al. Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev. 2001; 15: 3237-42.

[19] Grawunder U, Zimmer D, Fugmann S et al. DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. Mol Cell. 1998; 2: 477-84.

[20] Qi Y, Zhang Y, Zhang F et al. Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways. Genome Res. 2013; 23: 547-54.

[21] Grawunder U, Zimmer D, Kulesza P et al. Requirement for an interaction of XRCC4 with DNA ligase IV for wild-type V(D)J recombination and DNA double-strand break repair in vivo. J Biol Chem. 1998; 273: 24708-14.

[22] West CE, Waterworth WM, Jiang Q et al. Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4. Plant J. 2000; 24: 67-78.

[23] Chen XY, Wang P, Zhao F et al. The Liriodendron chinense MKK2 gene enhances Arabidopsis thaliana salt resistance. Forests 2020; 11: 1160.

[24] Mohanta TK, Arora PK, Mohanta N et al. Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants. BMC Genomics. 2015; 16: 58.

[25] Fan D, Liu T, Li C et al. Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation. Sci Rep. 2015; 5: 12217.

[26] Hsu PD, Scott DA, Weinstein JA et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013; 31: 827-32.

[27] Doench JG, Hartenian E, Graham DB et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol. 2014; 32: 1262-7.

[28] Song F, Stieger K . Optimizing the DNA donor template for homology-directed repair of double-strand breaks. Mol Ther Nucleic Acids. 2017; 7: 53-60.

[29] Gao J, Zhang S, He WD et al. Comparative phosphoproteomics reveals an important role of MKK2 in banana (Musa spp.) cold signal network. Sci Rep. 2017; 7: 40852.

[30] Hajiahmadi Z, Movahedi A, Wei H et al. Strategies to increase on-target and reduce off-target effects of the CRISPR/Cas9 system in plants. Int J Mol Sci. 2019; 20: E3719.

[31] Aslan Y, Tadjuidje E, Zorn AM et al. High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus. Development. 2017; 144: 2852-8.

[32] Danilo B, Perrot L, Botton E et al. The DFR locus. A smart landing pad for targeted transgene insertion in tomato. PLoS One 2018; 13: e0208395.

[33] Jang DE, Lee JY, Lee JH et al. Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency. Exp Mol Med. 2018; 50: 1-9.

[34] Menchaca A, dos Santos-Neto PC, Souza-Neves M et al. Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated homology directed repair. Sci Rep. 2020; 10: 5995.

[35] Liu T, Fan D, Ran LY et al. Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus. Hereditas (Beijing). 2015; 37: 1044-52.

[36] Shao SM, Ren C, Liu Z et al. Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52. Int J Biochem. 2017; 92: 43-52.

[37] Li GL, Liu D, Zhang X et al. Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts. Int J Biochem. 2018; 99: 154-60.

[38] Ceccaldi R, Rondinelli B, D’Andrea AD . Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 2016; 26: 52-64.

[39] Teige M, Scheikl E, Eulgem T et al. The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell. 2004; 15: 141-52.

[40] Sun W, Chen H, Wang J et al. Expression analysis of genes encoding mitogen-activated protein kinases in maize provides a key link between abiotic stress signaling and plant reproduction. Funct Integr Genomics. 2015; 15: 107-20.

[41] Zhang JP, Li XL, Li GH et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol. 2017; 18: 35.

[42] Xie KB, Yang YN . RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant. 2013; 6: 1975-83.

[43] Sambrook J, Fritsch EF, Maniatis T . Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.

[44] Sentmanat MF, Peters ST, Florian CP et al. A survey of validation strategies for CRISPR-Cas9 editing. Sci Rep 2018; 8: 888.