Editing Features of High-fidelity CRISPR/Cas9 System in Poultry Cells

doi:10.13560/j.cnki.biotech.bull.1985.2024-0673

Abstract

Abstract:

【Objective】 The emergence of high-fidelity editors in recent years is expected to widely reduce the off-target effect that occurs during gene editing in poultry with the conventional CRISPR/Cas9 system. However, there is still a lack of data support for the application of these high-fidelity editors in poultry. This study aims to further evaluate their on-target editing effects and off-target characteristics in poultry cells, so as to provide an effective reference for molecular genetic breeding and germplasm resource utilization in poultry.【Method】 Five high-fidelity Cas9 variants, which have been reported with high accuracy, were selected and co-transfected with the designed sgRNAs in chicken DF-1 cells, and the positive cells were collected for gene sequencing, and the editing efficiency and off-target effect of different high-fidelity editors were compared in parallel.【Result】 eCas9 maintained high editing activity while demonstrating low off-target effects in multiple sgRNAs tested against the avian influenza resistance-related gene ANP32B and the reproduction-related gene DAZL. Meanwhile, SuperFiCas9 had the lowest off-target effect among multiple tested high-fidelity variants, but a significant decrease in editing efficiency occurred in gene editing of poultry DF-1 cells.【Conclusion】 A variety of high-fidelity editors have shown editing activity in poultry cells, among which the eCas9 editor has high efficiency and low off-target properties, and can be used as a preferred high-fidelity editing tool for the genetic improvement of economically important traits and molecular breeding work in chickens.

Key words: chicken, high-fidelity editor, on-target, off-target, ANP32B

JIAO Dan-rong, MA Meng-xue, HE Bai-shui, XIE Long, ZUO Er-wei. Editing Features of High-fidelity CRISPR/Cas9 System in Poultry Cells[J]. Biotechnology Bulletin, 2024, 40(10): 191-197.

Figures/Tables 4

Table 1 Primer list

引物名称Primer name	sgRNA打靶位点sgRNA target-sites（5'-3'）	引物序列Primer sequence（5'-3'）	基因Gene
SgRNA1	ATAACTGCCGTTCAGATGAT	F：ATAACTGCCGTTCAGATGAT R：ATCATCTGAACGGCAGTTATC	ANP32B
SgRNA2	GGAGATTGGAGATGGACAGC	F：GAGATTGGAGATGGACAGC R：GCTGTCCATCTCCAATCTCC	ANP32B
SgRNA3	GTCCATCCCCATCTGCCTCA	F：TCCATCCCCATCTGCCTCA R：TGAGGCAGATGGGGATGGAC	ANP32B
SgRNA4	GGAAGAGGAGGATGATGATG	F：GAAGAGGAGGATGATGATG R：CATCATCATCCTCCTCTTCC	ANP32B
SgRNA5	TGATCTGGAAGGTGAAGAGG	F：TGATCTGGAAGGTGAAGAGG R：CCTCTTCACCTTCCAGATCAC	ANP32B
SgRNA6	TGAACAATATGGTACTGTGA	F：TGAACAATATGGTACTGTGA R：TCACAGTACCATATTGTTCAC	DAZL

Fig. 1 Construction process of plasmid sgRNA and mapping of different high-fidelity editors a: Construction process of sgRNA plasmid; b: the structure of plasmid editors

Fig. 2 Editing features of different high-fidelity editors in chicken gene ANP32B and DAZL a: The experiment schedule of gene editing in chicken cells for high-fidelity editors; b: editing efficiency comparing of different high-fidelity editors in different target sites from ANP32B and DAZL; c: detailed editing characteristics of different high-fidelity editors in ANP32B-site5 target site; d: the editing windows of different high-fidelity editors in ANP32B-site5

Fig. 3 Off-target editing of different high-fidelity editors in editing chicken's ANP32B and DAZL loci a: The off-target effects at ANP32B-site1, 3, 4 of different Cas9 variants; b: the detail off-target characteristics induced by different Cas9 varaints at ANP32B-site4-off7 locus; c: joint analysis of the on-target and off-target effects of Cas9 variants

References 25

[1]	邓蓉. 我国家禽产业现状分析及未来发展趋势[J]. 现代化农业, 2020(3): 68-71.
	Deng R. Present situation analysis and future development trend of poultry industry in China[J]. Mod Agric, 2020(3): 68-71.
[2]	刘旗. 我国畜禽遗传资源面临的问题及解决途径[J]. 农业技术与装备, 2012(15): 43-45.
	Liu Q. Problems and solutions of livestock and poultry genetic resources in China[J]. Agric Technol Equip, 2012(15): 43-45.
[3]	Zhang F. CRISPR-Cas9: prospects and challenges[J]. Hum Gene Ther, 2015, 26(7): 409-410. doi: 10.1089/hum.2015.29002.fzh pmid: 26176430
[4]	Fu YF, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J]. Nat Biotechnol, 2013, 31(9): 822-826. doi: 10.1038/nbt.2623 pmid: 23792628
[5]	Anderson KR, Haeussler M, Watanabe C, et al. CRISPR off-target analysis in genetically engineered rats and mice[J]. Nat Methods, 2018, 15(7): 512-514. doi: 10.1038/s41592-018-0011-5 pmid: 29786090
[6]	Zhang BY, Wang CY, Zhang Y, et al. A CRISPR-engineered swine model of COL2A1 deficiency recapitulates altered early skeletal developmental defects in humans[J]. Bone, 2020, 137: 115450.
[7]	Tang HH, Wang DQ, Shu YL. Structural insights into Cas9 mismatch: promising for development of high-fidelity Cas9 variants[J]. Sig Transduct Target Ther, 2022, 7: 271.
[8]	Vakulskas CA, Dever DP, Rettig GR, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells[J]. Nat Med, 2018, 24(8): 1216-1224. doi: 10.1038/s41591-018-0137-0 pmid: 30082871
[9]	Ikeda A, Fujii W, Sugiura K, et al. High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes[J]. Commun Biol, 2019, 2: 371. doi: 10.1038/s42003-019-0627-8 pmid: 31633062
[10]	Walton RT, Christie KA, Whittaker MN, et al. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants[J]. Science, 2020, 368(6488): 290-296. doi: 10.1126/science.aba8853 pmid: 32217751
[11]	Bravo JPK, Liu MS, Hibshman GN, et al. Structural basis for mismatch surveillance by CRISPR-Cas9[J]. Nature, 2022, 603(7900): 343-347.
[12]	Zong Y, Wang YP, Li C, et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion[J]. Nat Biotechnol, 2017, 35(5): 438-440. doi: 10.1038/nbt.3811 pmid: 28244994
[13]	Tang X, Ren QR, Yang LJ, et al. Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing[J]. Plant Biotechnol J, 2019, 17(7): 1431-1445. doi: 10.1111/pbi.13068 pmid: 30582653
[14]	Kim N, Choi S, Kim S, et al. Deep learning models to predict the editing efficiencies and outcomes of diverse base editors[J]. Nat Biotechnol, 2024, 42(3): 484-497.
[15]	Idoko-Akoh A, Goldhill DH, Sheppard CM, et al. Creating resistance to avian influenza infection through genome editing of the ANP32 gene family[J]. Nat Commun, 2023, 14(1): 6136. doi: 10.1038/s41467-023-41476-3 pmid: 37816720
[16]	Rengaraj D, Zheng YH, Kang KS, et al. Conserved expression pattern of chicken DAZL in primordial germ cells and germ-line cells[J]. Theriogenology, 2010, 74(5): 765-776. doi: 10.1016/j.theriogenology.2010.04.001 pmid: 20537692
[17]	Kulcsár PI, Tálas A, Huszár K, et al. Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage[J]. Genome Biol, 2017, 18(1): 190. doi: 10.1186/s13059-017-1318-8 pmid: 28985763
[18]	Hu JH, Miller SM, Geurts MH, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity[J]. Nature, 2018, 556(7699): 57-63.
[19]	Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease[J]. Nature, 2014, 513(7519): 569-573.
[20]	Zhang DB, Zhang HW, Li TD, et al. Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases[J]. Genome Biol, 2017, 18(1): 191. doi: 10.1186/s13059-017-1325-9 pmid: 29020979
[21]	戴小芳. 鸡病流行特点及防治措施分析[J]. 新农民, 2024(11):126-128.
	Dai XF. Analysis of epidemic characteristics and preventive measures of chicken diseases[J]. New Farmer, 2024(11):126-128.
[22]	张迎冰, 张成图, 吴英, 等. 单碱基编辑技术的原理、发展及其在家畜育种中的应用[J]. 生物工程学报, 2023, 39(1):19-33.
	Zhang YB, Zhang CT, Wu Y, et al. Principles and development of single base editing technology and its application in livestock breeding[J]. Journal of Biological Engineering, 2023, 39(1):19-33.
[23]	Chen JS, Dagdas YS, Kleinstiver BP, et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy[J]. Nature, 2017, 550(7676): 407-410.
[24]	Kim N, Kim HK, Lee S, et al. Prediction of the sequence-specific cleavage activity of Cas9 variants[J]. Nat Biotechnol, 2020, 38(11): 1328-1336.
[25]	Kulcsár PI, Tálas A, Ligeti Z, et al. SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e[J]. Nat Commun, 2022, 13(1): 6858. doi: 10.1038/s41467-022-34527-8 pmid: 36369279