引导编辑技术的研究进展及应用

doi:10.11843/j.issn.0366-6964.2024.04.001

摘要/Abstract

摘要： 以CRISPR/Cas9为基础的引导编辑系统是新开发出的一种基因编辑技术，可以精确实现12种碱基的互换、插入和缺失，并且不需要产生双链断裂和引入外源供体DNA。本文从基因编辑的发展、引导编辑系统的原理、特点、优化、脱靶效应、在动植物和基因治疗研究中的应用、引导编辑系统的设计等几方面进行综述，为促进相关领域的科研工作者了解引导编辑系统及进一步利用引导编辑系统在动植物科学基础研究和育种方面的应用提供指导。

关键词: CRISPR/Cas9, 引导编辑(prime editing, PE), pegRNA, 应用研究

Abstract: The prime editing system based on CRISPR/Cas9 is a novel gene editing technology, which enables all 12 base-to-base conversions or transition, precise small insertion and deletion without requiring double-strand breaks or exogenous donor DNA templates. This review summarizes the development of gene editing approaches, the principle and characteristics of prime editing technique, as well as applications in animal and plant breeding and human gene therapy. It provides a reference for scientists to get insight into prime editing system and further use it in basic research and animal or plant breeding.

Key words: CRISPR/Cas9, prime editing(PE), pegRNA, application research

中图分类号:

S813.1

邱梅玉, 张雪梅, 张宁, 刘明军. 引导编辑技术的研究进展及应用[J]. 畜牧兽医学报, 2024, 55(4): 1345-1355.

QIU Meiyu, ZHANG Xuemei, ZHANG Ning, LIU Mingjun. Approach and Application of Prime Editing System[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1345-1355.

参考文献

[1] LIU S J, DUO S G. Research progress and application of CRISPR/Cas gene editing technology[J]. Journal of Inner Mongolia University:Natural Science Edition, 2019, 50(5):557-563. (in Chinese)
刘树君, 多曙光. CRISPR/Cas基因编辑系统的研究进展及应用[J]. 内蒙古大学学报:自然科学版, 2019, 50(5):557-563.
[2] CECCALDI R, RONDINELLI B, D'ANDREA A D. Repair pathway choices and consequences at the double-strand break[J]. Trends Cell Biol, 2016, 26(1):52-64.
[3] PAQUET D, KWART D, CHEN A, et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9[J]. Nature, 2016, 533(7601):125-129.
[4] XU X, LIU M J. Recent advances and applications of base editing systems[J]. Chinese Journal of Biotechnology, 2021, 37(7):2307-2321. (in Chinese)
徐鑫, 刘明军. 碱基编辑系统研究最新进展及应用[J]. 生物工程学报, 2021, 37(7):2307-2321.
[5] KOMOR A C, KIM Y B, PACKER M S, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603):420-424.
[6] GAUDELLI N M, KOMOR A C, REES H A, et al. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage[J]. Nature, 2017, 551(7681):464-471.
[7] ANZALONE A V, RANDOLPH P B, DAVIS J R, et al. Search-and-replace genome editing without double-strand breaks or donor DNA[J]. Nature, 2019, 576(7785):149-157.
[8] LIU Y, HUANG X X, WANG X L. Search-and-replace editing of genetic information[J]. Front Agric Sci Eng, 2020, 7(2):231-232.
[9] DU Q L, WANG C, LIU G W, et al. Plant prime editing technique:a new genome editing tool for plants[J]. Chinese Journal of Biotechnology, 2022, 38(1):26-33. (in Chinese)
杜秋丽, 王超, 刘关稳, 等. 植物基因组编辑新工具——引导编辑技术[J]. 生物工程学报, 2022, 38(1):26-33.
[10] QIN R Y, WEI P C. Prime editing creates a novel dimension of plant precise genome editing[J]. Hereditas (Beijing), 2020, 42(6):519-523. (in Chinese)
秦瑞英, 魏鹏程. Prime editing引导植物基因组精确编辑新局面[J]. 遗传, 2020, 42(6):519-523.
[11] YAN J, CIRINCIONE A, ADAMSON B. Prime editing:precision genome editing by reverse transcription[J]. Mol Cell, 2020, 77(2):210-212.
[12] LIU Y, ZHOU X H, HUANG S H, et al. Prime editing:a search and replace tool with versatile base changes[J]. Hereditas (Beijing), 2022, 44(11):993-1008. (in Chinese)
刘尧, 周先辉, 黄舒泓, 等. 引导编辑:突破碱基编辑类型的新技术[J]. 遗传, 2022, 44(11):993-1008.
[13] LIU P P, LIANG S Q, ZHENG C W, et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice[J]. Nat Commun, 2021, 12(1):2121.
[14] XU W, ZHANG C W, YANG Y X, et al. Versatile nucleotides substitution in plant using an improved prime editing system[J]. Mol Plant, 2020, 13(5):675-678.
[15] XU W, YANG Y X, YANG B Y, et al. A design optimized prime editor with expanded scope and capability in plants[J]. Nat Plants, 2022, 8(1):45-52.
[16] LU Y M, TIAN Y F, SHEN R D, et al. Precise genome modification in tomato using an improved prime editing system[J]. Plant Biotechnol J, 2021, 19(3):415-417.
[17] CHEN P J, HUSSMANN J A, YAN J, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes[J]. Cell, 2021, 184(22):5635-5652.e29.
[18] ZHANG G Q, LIU Y, HUANG S S, et al. Enhancement of prime editing via xrRNA motif-joined pegRNA[J]. Nat Commun, 2022, 13(1):1856.
[19] LIU Y, YANG G, HUANG S H, et al. Enhancing prime editing by Csy4-mediated processing of pegRNA[J]. Cell Res, 2021, 31(10):1134-1136.
[20] FERREIRA DA SILVA J, OLIVEIRA G P, ARASA-VERGE E A, et al. Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair[J]. Nat Commun, 2022, 13(1):760.
[21] JIANG T T, ZHANG X O, WENG Z P, et al. Deletion and replacement of long genomic sequences using prime editing[J]. Nat Biotechnol, 2022, 40(2):227-234.
[22] CHOI J, CHEN W, SUITER C C, et al. Precise genomic deletions using paired prime editing[J]. Nat Biotechnol, 2022, 40(2):218-226.
[23] ANZALONE A V, GAO X D, PODRACKY C J, et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing[J]. Nat Biotechnol, 2022, 40(5):731-740.
[24] WANG J L, HE Z, WANG G Q, et al. Efficient targeted insertion of large DNA fragments without DNA donors[J]. Nat Methods, 2022, 19(3):331-340.
[25] LI X Y, WANG X, SUN W J, et al. Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes[J]. J Mol Cell Biol, 2022, 14(4):mjac022.
[26] NELSON J W, RANDOLPH P B, SHEN S P, et al. Engineered pegRNAs improve prime editing efficiency[J]. Nat Biotechnol, 2022, 40(3):402-410.
[27] LI X S, ZHOU L N, GAO B Q, et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure[J]. Nat Commun, 2022, 13(1):1669.
[28] LIU B, DONG X L, CHENG H Y, et al. A split prime editor with untethered reverse transcriptase and circular RNA template[J]. Nat Biotechnol, 2022, 40(9):1388-1393.
[29] FENG Y, LIU S Y, MO Q Q, et al. Enhancing prime editing efficiency and flexibility with tethered and split pegRNAs[J]. Protein Cell, 2023, 14(4):304-308.
[30] LIN Q P, JIN S, ZONG Y, et al. High-efficiency prime editing with optimized, paired pegRNAs in plants[J]. Nat Biotechnol, 2021, 39(8):923-927.
[31] WOLFF J H, HALDRUP J, THOMSEN E A, et al. piggyPrime:high-efficacy prime editing in human cells using piggyBac-based DNA transposition[J]. Front Appl Math Stat, 2021:3:786893.
[32] EGGENSCHWILER R, GSCHWENDTBERGER T, FELSKI C, et al. A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines[J]. Sci Rep, 2021, 11(1):22154.
[33] WANG Q, LIU J, JANSSEN J M, et al. Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery[J]. Nucleic Acids Res, 2021, 49(20):11986-12001.
[34] ADIKUSUMA F, LUSHINGTON C, ARUDKUMAR J, et al. Optimized nickase- and nuclease-based prime editing in human and mouse cells[J]. Nucleic Acids Res, 2021, 49(18):10785-10795.
[35] XU R F, LI J, LIU X S, et al. Development of plant prime-editing systems for precise genome editing[J]. Plant Commun, 2020, 1(3):100043.
[36] SIMON D A, TÁLAS A, KULCSÁR P I, et al. PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells[J]. eLife, 2022, 11:e69504.
[37] SCHENE I F, JOORE I P, BAIJENS J H L, et al. Mutation-specific reporter for optimization and enrichment of prime editing[J]. Nat Commun, 2022, 13(1):1028.
[38] OH Y, LEE W J, HUR J K, et al. Expansion of the prime editing modality with Cas9 from Francisella novicida[J]. Genome Biol, 2022, 23(1):92.
[39] SCHENE I F, JOORE I P, OKA R, et al. Prime editing for functional repair in patient-derived disease models[J]. Nat Commun, 2020, 11(1):5352.
[40] ZONG Y, LIU Y J, XUE C X, et al. An engineered prime editor with enhanced editing efficiency in plants[J]. Nat Biotechnol, 2022, 40(9):1394-1402.
[41] NELSON J W, RANDOLPH P B, SHEN S P, et al. Engineered pegRNAs improve prime editing efficiency[J]. Nat Biotechnol, 2022, 40(3):402-410.
[42] GAO R Z, FU Z C, LI X Y, et al. Genomic and transcriptomic analyses of prime editing guide RNA-independent off-target effects by prime editors[J]. CRISPR J, 2022, 5(2):276-293.
[43] GEURTS M H, DE POEL E, PLEGUEZUELOS-MANZANO C, et al. Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids[J]. Life Sci Alliance, 2021, 4(10):e202000940.
[44] HABIB O, HABIB G, HWANG G H, et al. Comprehensive analysis of prime editing outcomes in human embryonic stem cells[J]. Nucleic Acids Res, 2022, 50(2):1187-1197.
[45] PARK S J, JEONG T Y, SHIN S K, et al. Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor[J]. Genome Biol, 2021, 22(1):170.
[46] GAO P, LYU Q, GHANAM A R, et al. Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression[J]. Genome Biol, 2021, 22(1):83.
[47] BÖCK D, ROTHGANGL T, VILLIGER L, et al. In vivo prime editing of a metabolic liver disease in mice[J]. Sci Transl Med, 2022, 14(636):eabl9238.
[48] QIAN Y Q, ZHAO D, SUI T, et al. Efficient and precise generation of Tay-Sachs disease model in rabbit by prime editing system[J]. Cell Discov, 2021, 7(1):50.
[49] KIM D E, LEE J H, JI K B, et al. Prime editor-mediated correction of a pathogenic mutation in purebred dogs[J]. Sci Rep, 2022, 12(1):12905.
[50] LIU Y, LI X Y, HE S T, et al. Efficient generation of mouse models with the prime editing system[J]. Cell Discov, 2020, 6:27.
[51] PETRI K, ZHANG W T, MA J Y, et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells[J]. Nat Biotechnol, 2022, 40(2):189-193.
[52] BOSCH J A, BIRCHAK G, PERRIMON N. Precise genome engineering in Drosophila using prime editing[J]. Proc Natl Acad Sci U S A, 2021, 118(1):e2021996118.
[53] ATSUTA Y, SUZUKI K, IIKAWA H, et al. Prime editing in chicken fibroblasts and primordial germ cells[J]. Dev Growth Differ, 2022, 64(9):548-557.
[54] AIDA T, WILDE J J, YANG L X, et al. Prime editing primarily induces undesired outcomes in mice[J]. bioRxiv, 2020.
[55] TANG X, SRETENOVIC S, REN Q R, et al. Plant prime editors enable precise gene editing in rice cells[J]. Mol Plant, 2020, 13(5):667-670.
[56] HUA K, JIANG Y W, TAO X P, et al. Precision genome engineering in rice using prime editing system[J]. Plant Biotechnol J, 2020, 18(11):2167-2169.
[57] LIN Q P, ZONG Y, XUE C X, et al. Prime genome editing in rice and wheat[J]. Nat Biotechnol, 2020, 38(5):582-585.
[58] XU R F, LIU X S, LI J, et al. Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice[J]. Nat Plants, 2021, 7(7):888-892.
[59] BISWAS S, BRIDGELAND A, IRUM S, et al. Optimization of prime editing in rice, peanut, chickpea, and cowpea protoplasts by restoration of GFP activity[J]. Int J Mol Sci, 2022, 23(17):9809.
[60] JIANG Y Y, CHAI Y P, LU M H, et al. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize[J]. Genome Biol, 2020, 21(1):257.
[61] NIU X R, YIN S M, CHEN X, et al. Gene editing technology and its recent progress in disease therapy[J]. Hereditas (Beijing), 2019, 41(7):582-598. (in Chinese)
牛煦然, 尹树明, 陈曦, 等. 基因编辑技术及其在疾病治疗中的研究进展[J]. 遗传, 2019, 41(7):582-598.
[62] SCHENE I F, JOORE I P, OKA R, et al. Prime editing for functional repair in patient-derived disease models[J]. Nat Commun, 2020, 11(1):5352.
[63] ZHOU M J, TANG S Q, DUAN N N, et al. Targeted-deletion of a tiny sequence via prime editing to restore SMN expression[J]. Int J Mol Sci, 2022, 23(14):7941.
[64] CHEMELLO F, CHAI A C, LI H, et al. Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing[J]. Sci Adv, 2021, 7(18):eabg4910.
[65] JANG H, JO D H, CHO C S, et al. Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases[J]. Nat Biomed Eng, 2022, 6(2):181-194.
[66] KIM Y, HONG S A, YU J, et al. Adenine base editing and prime editing of chemically derived hepatic progenitors rescue genetic liver disease[J]. Cell Stem Cell, 2021, 28(9):1614-1624.e5.
[67] LI C, GEORGAKOPOULOU A, NEWBY G A, et al. In vivo HSC prime editing rescues sickle cell disease in a mouse model[J]. Blood, 2023, 141(17):2085-2099.
[68] HSU J Y, GRVNEWALD J, SZALAY R, et al. PrimeDesign software for rapid and simplified design of prime editing guide RNAs[J]. Nat Commun, 2021, 12(1):1034.
[69] CHOW R D, CHEN J S, SHEN J, et al. A web tool for the design of prime-editing guide RNAs[J]. Nat Biomed Eng, 2021, 5(2):190-194.
[70] ANDERSON M V, HALDRUP J, THOMSEN E A, et al. pegIT-a web-based design tool for prime editing[J]. Nucleic Acids Res, 2021, 49(W1):W505-W509.
[71] HWANG G H, JEONG Y K, HABIB O, et al. PE-Designer and PE-Analyzer:web-based design and analysis tools for CRISPR prime editing[J]. Nucleic Acids Res, 2021, 49(W1):W499-W504.
[72] STANDAGE-BEIER K, TEKEL S J, BRAFMAN D A, et al. Prime editing guide RNA design automation using PINE-CONE[J]. ACS Synth Biol, 2021, 10(2):422-427.
[73] SIEGNER S M, KARASU M E, SCHRÖDER M S, et al. PnB Designer:a web application to design prime and base editor guide RNAs for animals and plants[J]. BMC Bioinformatics, 2021, 22(1):101.
[74] LI Y C, CHEN J J, TSAI S Q, et al. Easy-Prime:a machine learning-based prime editor design tool[J]. Genome Biol, 2021, 22(1):235.
[75] KOBLAN L W, DOMAN J L, WILSON C, et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction[J]. Nat Biotechnol, 2018, 36(9):843-846.
[76] FU J H, LI Q, LIU X Y, et al. Human cell based directed evolution of adenine base editors with improved efficiency[J]. Nat Commun, 2021, 12(1):5897.
[77] LANDRUM M J, LEE J M, BENSON M, et al. ClinVar:public archive of interpretations of clinically relevant variants[J]. Nucleic Acids Res, 2016, 44(D1):D862-D868.
[78] ZHENG C W, LIANG S Q, LIU B, et al. A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver[J]. Mol Ther, 2022, 30(3):1343-1351.