非编码RNA的引导RNA的设计及其应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0750

摘要/Abstract

摘要：

非编码RNA（non-coding RNA, ncRNA）是指通常不编码蛋白质的功能性RNA分子，多通过调控编码基因的表达和功能发挥重要的生理作用。微小RNA（microRNA, miRNA）、环状RNA（circular RNA, circRNA）及长编码RNA（long non-coding RNA, lncRNA）是目前已知的三大类具有重要生理和病理意义的调控型ncRNA，其基因的缺失重排和表达失调与多种疾病的发生发展密切相关。成簇规律间隔短回文重复序列及相关蛋白质系统（clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins, CRISPR/Cas）是一类广泛存在于细菌和古菌中的免疫防御系统，由CRISPR相关蛋白（CRISPR-associated proteins, Cas）和引导RNA（guide RNAs, gRNA）共同组成。该系统利用gRNA引导Cas核酸酶剪切和清除外源基因，因此Cas酶对靶DNA/RNA的精准识别和有效切割是由gRNA决定的。CRISPR/Cas技术具有优异的基因组定点编辑和转录本特异性剪切能力，能精准高效地编辑、干预和检测ncRNA的异常，帮助人们成功诊治疾病。本文在总结Cas9和Cas13 gRNA的组成和设计原则的基础上，针对miRNA、circRNA及lncRNA基因和其RNA自身或RNA形成过程中产生的特殊分子特征，重点介绍针对三类ncRNA的gRNA的设计策略和敲除靶点的选择，以及Cas9和Cas13核酸酶如何在gRNA高特异性和高效率的引导下，对异常miRNA、circRNA和lncRNA进行精准检测和干预。进一步讨论3类ncRNA的gRNA设计异同点和CRISRP/Cas应用在ncRNA上所面临的挑战，并对未来精准靶向ncRNA的gRNA设计进行展望，旨在为CRISPR/Cas技术在治疗ncRNA相关疾病上的应用提供参考。

关键词: CRISPR/Cas, guide RNA, 非编码RNA, miRNAs, circRNAs, lncRNAs, 敲除靶点

Abstract:

Non-coding RNA(ncRNA) refers to functional RNA molecules that typically do not encode proteins but play important physiological roles by regulating the expression and function of coding genes. microRNA (miRNA), circular RNA (circRNA), and long non-coding RNA (lncRNA) are currently known as the three major types of regulatory ncRNAs with important physiological and pathological significance. The loss, rearrangement, and dysregulation of their genes are closely related to the occurrence and development of various diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas) are types of immune-defense systems widely existing in bacteria and archaea, consisting of CRISPR-associated proteins (Cas) and guide RNAs (gRNAs). Cas nucleases shear and remove exogenous genes, while gRNAs determine their precise recognition and effective cleavage of target DNA/RNAs. CRISPR/Cas technology has excellent capabilities of site-specific genome editing and cleavage of RNA transcripts and can precisely and efficiently edit, intervene, and detect abnormalities in these ncRNAs and thus contribute to successful diagnosis and treatment of diseases. This paper summarizes the composition and design principles of Cas9 and Cas13 gRNA and the unique molecular characteristics of miRNA, circRNA, and lncRNA genes and their respective molecular features produced by the RNA itself or during RNA formation, emphasizes the design strategies and target selection of their gRNAs, as well as how Cas9 and Cas13 nucleases can accurately detect and intervene in abnormal miRNA, circRNA, and lncRNA under the guidance of gRNA with high specificity and efficiency. The article further discusses the similarities and differences in gRNA design among three types of ncRNAs, as well as the challenges faced by CRISPR/Cas applications in ncRNAs. It also gives prospects for future gRNA designs for more accurate targeting at ncRNAs, aiming to provide some reference for the employment of CRISPR/Cas technology in managing ncRNAs-associated diseases.

Key words: CRISPR/Cas, guide RNA, non-coding RNA, miRNAs, circRNAs, lncRNAs, knockdown targets

秦玉婷, 潘森涛, 陈渝萍. 非编码RNA的引导RNA的设计及其应用[J]. 生物技术通报, 2025, 41(3): 71-82.

QIN Yu-ting, PAN Sen-tao, CHEN Yu-ping. Design and Application of Guide RNAs for Non-coding RNAs[J]. Biotechnology Bulletin, 2025, 41(3): 71-82.

图/表 4

参考文献 76

1	Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system [J]. Cell, 2015, 163(3): 759-771.
2	Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity [J]. Science, 2012, 337(6096): 816-821.
3	Kordyś M, Sen R, Warkocki Z. Applications of the versatile CRISPR-Cas13 RNA targeting system [J]. Wiley Interdiscip Rev RNA, 2022, 13(3): e1694.
4	Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies [J]. Cell, 2024, 187(5): 1076-1100.
5	Feng Q, Li QR, Zhou HZ, et al. CRISPR technology in human diseases [J]. MedComm, 2024, 5(8): e672.
6	Granados-Riveron JT, Aquino-Jarquin G. CRISPR/Cas13-based approaches for ultrasensitive and specific detection of microRNAs [J]. Cells, 2021, 10(7): 1655.
7	Matsui M, Corey DR. Non-coding RNAs as drug targets [J]. Nat Rev Drug Discov, 2017, 16(3): 167-179.
8	谭璐媛, 王妍, 钟文静, 等. 非编码RNA作为疾病诊断标志物 [J]. 生命科学, 2018, 30(2): 204-212.
	Tan LY, Wang Y, Zhong WJ, et al. Non-coding RNA biomarkers for diagnosis of human diseases [J]. Chin Bull Life Sci, 2018, 30(2): 204-212.
9	Calin GA, Liu CG, Ferracin M, et al. Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas [J]. Cancer Cell, 2007, 12(3): 215-229.
10	Slack FJ, Chinnaiyan AM. The role of non-coding RNAs in oncology [J]. Cell, 2019, 179(5): 1033-1055.
11	Borkiewicz L, Kalafut J, Dudziak K, et al. Decoding LncRNAs [J]. Cancers: Basel, 2021, 13(11): 2643.
12	Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and Archaea [J]. Nature, 2012, 482(7385): 331-338.
13	Jiang FG, Doudna JA. CRISPR-Cas9 structures and mechanisms [J]. Annu Rev Biophys, 2017, 46: 505-529.
14	李江, 耿立召, 许建平. CRISPR/Cas9系统中引导RNA的研究进展 [J]. 生物技术通报, 2019, 35(4): 108-115.
	Li J, Geng LZ, Xu JP. Research progress on guide RNA in CRISPR/Cas9 system [J]. Biotechnol Bull, 2019, 35(4): 108-115.
15	陈静, 赵燕波, 赵兰, 等. 靶向RNA的CRISPR-Cas13系统及其研究进展 [J]. 福建师范大学学报: 自然科学版, 2021, 37(2): 10-17.
	Chen J, Zhao YB, Zhao L, et al. RNA-targeting CRISPR-Cas13 systems and its research progress [J]. J Fujian Norm Univ Nat Sci Ed, 2021, 37(2): 10-17.
16	Mohr SE, Hu YH, Ewen-Campen B, et al. CRISPR guide RNA design for research applications [J]. FEBS J, 2016, 283(17): 3232-3238.
17	Zhang C, Konermann S, Brideau NJ, et al. Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d [J]. Cell, 2018, 175(1): 212-223.e17.
18	Zhang B, Ye YM, Ye WW, et al. Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d [J]. Nat Commun, 2019, 10(1): 2544.
19	Wessels HH, Méndez-Mancilla A, Guo XY, et al. Massively parallel Cas13 screens reveal principles for guide RNA design [J]. Nat Biotechnol, 2020, 38(6): 722-727.
20	Bandaru S, Tsuji MH, Shimizu Y, et al. Structure-based design of gRNA for Cas13 [J]. Sci Rep, 2020, 10(1): 11610.
21	Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases [J]. Nat Biotechnol, 2013, 31(9): 827-832.
22	Jonas S, Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing [J]. Nat Rev Genet, 2015, 16(7): 421-433.
23	Boudoukha S, Rivera Vargas T, Dang I, et al. MiRNA let-7g regulates skeletal myoblast motility via Pinch-2 [J]. FEBS Lett, 2014, 588(9): 1623-1629.
24	Liu XW, Yang S, Wang L, et al. Hierarchically tumor-activated nanoCRISPR-Cas13a facilitates efficient microRNA disruption for multi-pathway-mediated tumor suppression [J]. Theranostics, 2023, 13(9): 2774-2786.
25	Hong JS, Son T, Castro CM, et al. CRISPR/Cas13a-based microRNA detection in tumor-derived extracellular vesicles [J]. Adv Sci, 2023, 10(24): e2301766.
26	Jiang L, Du JL, Xu HL, et al. Ultrasensitive CRISPR/Cas13a-mediated photoelectrochemical biosensors for specific and direct assay of miRNA-21 [J]. Anal Chem, 2023, 95(2): 1193-1200.
27	Li JW, Chen C, Luo F, et al. Highly sensitive biosensor for specific miRNA detection based on cascade signal amplification and magnetic electrochemiluminescence nanoparticles [J]. Anal Chim Acta, 2024, 1288: 342123.
28	Ma XW, Zhou F, Yang DL, et al. miRNA detection for prostate cancer diagnosis by miRoll-cas: miRNA rolling circle transcription for CRISPR-cas assay [J]. Anal Chem, 2023, 95(35): 13220-13226.
29	Wang B, Xu YT, Zhang TY, et al. An ultrasensitive and efficient microRNA nanosensor empowered by the CRISPR/cas confined in a nanopore [J]. Nano Lett, 2024, 24(1): 202-208.
30	Jiang Q, Meng X, Meng LW, et al. Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance [J]. RNA Biol, 2014, 11(10): 1243-1249.
31	Chang H, Yi B, Ma RX, et al. CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo [J]. Sci Rep, 2016, 6: 22312.
32	冯万有, 杨燕燕, 蒙丽娜, 等. CRISPR/Cas9系统敲除miRNA-182和miRNA-155的初步研究 [J]. 畜牧兽医杂志, 2022, 41(2): 15-21.
	Feng WY, Yang YY, Meng LN, et al. Preliminary study on knockout of miRNA-182 and miRNA-155 by CRISPR/Cas9 system [J]. J Anim Sci Vet Med, 2022, 41(2): 15-21.
33	Christie KA, Guo JA, Silverstein RA, et al. Precise DNA cleavage using CRISPR-SpRYgests [J]. Nat Biotechnol, 2023, 41(3): 409-416.
34	Li MH, Liu XY, Dai SF, et al. High efficiency targeting of non-coding sequences using CRISPR/Cas9 system in Tilapia [J]. G3 (Bethesda), 2019, 9(1): 287-295.
35	Tian T, Shu BW, Jiang YZ, et al. An ultralocalized Cas13a assay enables universal and nucleic acid amplification-free single-molecule RNA diagnostics [J]. ACS Nano, 2021, 15(1): 1167-1178.
36	Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2 [J]. Science, 2017, 356(6336): 438-442.
37	Kellner MJ, Koob JG, Gootenberg JS, et al. SHERLOCK: nucleic acid detection with CRISPR nucleases [J]. Nat Protoc, 2019, 14(10): 2986-3012.
38	Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector [J]. Science, 2016, 353(6299): aaf5573.
39	Chen Y, Zhang YB, Luo SY, et al. Foldback-crRNA-enhanced CRISPR/Cas13a system (FCECas13a) enables direct detection of ultrashort sncRNA [J]. Anal Chem, 2023, 95(42): 15606-15613.
40	Kristensen LS, Andersen MS, Stagsted LVW, et al. The biogenesis, biology and characterization of circular RNAs [J]. Nat Rev Genet, 2019, 20(11): 675-691.
41	Dong XJ, Bai YF, Liao ZX, et al. Circular RNAs in the human brain are tailored to neuron identity and neuropsychiatric disease [J]. Nat Commun, 2023, 14(1): 5327.
42	He ZL, Zhu QB. Circular RNAs: Emerging roles and new insights in human cancers [J]. Biomed Pharmacother, 2023, 165: 115217.
43	Wu MC, Xun M, Chen YP. Circular RNAs: regulators of vascular smooth muscle cells in cardiovascular diseases [J]. J Mol Med, 2022, 100(4): 519-535.
44	Li X, Kang XL, Di YL, et al. CircCHMP5 contributes to O _x -LDL-induced endothelial cell injury through the regulation of miR-532-5p/ROCK2 axis [J]. Cardiovasc Drugs Ther, 2023, 37(6): 1-12.
45	Miao RY, Qi CR, Fu YQ, et al. Silencing of circARHGAP12 inhibits the progression of atherosclerosis via miR-630/EZH2/TIMP2 signal axis [J]. J Cell Physiol, 2022, 237(1): 1057-1069.
46	Li SQ, Li X, Xue W, et al. Screening for functional circular RNAs using the CRISPR-Cas13 system [J]. Nat Methods, 2021, 18(1): 51-59.
47	Zhang Y, Xue W, Li X, et al. The biogenesis of nascent circular RNAs [J]. Cell Rep, 2016, 15(3): 611-624.
48	Zhong J, Wu XJ, Gao YX, et al. Circular RNA encoded MET variant promotes glioblastoma tumorigenesis [J]. Nat Commun, 2023, 14(1): 4467.
49	Heumüller AW, Jones AN, Mourão A, et al. Locus-conserved circular RNA cZNF292 controls endothelial cell flow responses [J]. Circ Res, 2022, 130(1): 67-79.
50	Zheng QP, Bao CY, Guo WJ, et al. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs [J]. Nat Commun, 2016, 7: 11215.
51	He AT, Liu JL, Li FY, et al. Targeting circular RNAs as a therapeutic approach: current strategies and challenges [J]. Signal Transduct Target Ther, 2021, 6(1): 185.
52	Feng XY, Zhu SX, Pu KJ, et al. New insight into circRNAs: characterization, strategies, and biomedical applications [J]. Exp Hematol Oncol, 2023, 12(1): 91.
53	Ishola AA, Chien CS, Yang YP, et al. Oncogenic circRNA C190 promotes non-small cell lung cancer via modulation of the EGFR/ERK pathway [J]. Cancer Res, 2022, 82(1): 75-89.
54	Zhang Y, Nguyen TM, Zhang XO, et al. Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs [J]. Genome Biol, 2021, 22(1): 41.
55	Gomes-Duarte A, Venø MT, de Wit M, et al. Expression of Circ_Satb1 is decreased in mesial temporal lobe epilepsy and regulates dendritic spine morphology [J]. Front Mol Neurosci, 2022, 15: 832133.
56	Guttman M, Amit I, Garber M, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals [J]. Nature, 2009, 458(7235): 223-227.
57	Singh N. Role of mammalian long non-coding RNAs in normal and neuro oncological disorders [J]. Genomics, 2021, 113(5): 3250-3273.
58	Coan M, Haefliger S, Ounzain S, et al. Targeting and engineering long non-coding RNAs for cancer therapy [J]. Nat Rev Genet, 2024, 25(8): 578-595.
59	Xun M, Zhang J, Wu MC, et al. Long non-coding RNAs: the growth controller of vascular smooth muscle cells in cardiovascular diseases [J]. Int J Biochem Cell Biol, 2023, 157: 106392.
60	Zhang J, Luo QZ, Li X, et al. Novel role of immune-related non-coding RNAs as potential biomarkers regulating tumour immunoresponse via MICA/NKG2D pathway [J]. Biomark Res, 2023, 11(1): 86.
61	Moradi Z, Kazemi M, Jamshidi-Khalifelou R, et al. CRISPR du-HITI an attractive approach to targeting Long Noncoding RNA HCP5 as inhibitory factor for proliferation of ovarian cancer cell [J]. Funct Integr Genomics, 2024, 24(2): 61.
62	Zheng C, Wei Y, Zhang P, et al. CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer [J]. J Clin Invest, 2023, 133(5): e159940.
63	Chan YT, Wu JY, Lu YJ, et al. Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC [J]. Mol Cancer, 2024, 23(1): 74.
64	Wang L, Bitar M, Lu X, et al. CRISPR-Cas13d screens identify KILR, a breast cancer risk-associated lncRNA that regulates DNA replication and repair [J]. Mol Cancer, 2024, 23(1): 101.
65	Zhu SY, Li W, Liu JZ, et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library [J]. Nat Biotechnol, 2016, 34(12): 1279-1286.
66	Arnan C, Ullrich S, Pulido-Quetglas C, et al. Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages [J]. BMC Genomics, 2022, 23(1): 402.
67	Zibitt MS, Hartford CCR, Lal A. Interrogating lncRNA functions via CRISPR/Cas systems [J]. RNA Biol, 2021, 18(12): 2097-2106.
68	Goyal A, Myacheva K, Groß M, et al. Challenges of CRISPR/Cas9 applications for long non-coding RNA genes [J]. Nucleic Acids Res, 2017, 45(3): e12.
69	Gilbert LA, Horlbeck MA, Adamson B, et al. Genome-scale CRISPR-mediated control of gene repression and activation [J]. Cell, 2014, 159(3): 647-661.
70	Liu SJ, Horlbeck MA, Cho SW, et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells [J]. Science, 2017, 355(6320): aah7111.
71	Liu Y, Cao ZZ, Wang YN, et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites [J]. Nat Biotechnol, 2018: 36(12):1203-1210.
72	Horlbeck MA, Liu SJ, Chang HY, et al. Fitness effects of CRISPR/Cas9-targeting of long noncoding RNA genes [J]. Nat Biotechnol, 2020, 38(5): 573-576.
73	Zhang SK, Chen Y, Dong KZ, et al. BESST: a novel lncRNA knockout strategy with less genome perturbance [J]. Nucleic Acids Res, 2023, 51(9): e49.
74	Yang ZT, Zhang ZX, Li J, et al. CRISPRlnc: a machine learning method for lncRNA-specific single-guide RNA design of CRISPR/Cas9 system [J]. Brief Bioinform, 2024, 25(2): bbae066.
75	Xu DY, Cai Y, Tang L, et al. A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response [J]. Sci Rep, 2020, 10(1): 1794.
76	Montero JJ, Trozzo R, Sugden M, et al. Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx [J]. Nat Methods, 2024, 21(4): 584-596.

编辑推荐 0

Metrics

阅读次数

全文

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	2	0	0	12

来源	本网站	其他网站

次数	14	0
比例	100%	0%

摘要

最新录用	在线预览	正式出版

0	0	30

	来源	本网站

	次数	30
	比例	100%

工具 Tool	输入 Input	网址 Web site
CHOPCHOP	DNA 序列；基因名称；基因组位置	https：//chopchop.cbu.uib.no
CRISPRDB	基因名称	https：//crisprdb.org
CRISPRscan	DNA 序列	https：//www.crisprscan.org
Synthego	基因名称	https：//design.synthego.com
EuPaGDT	DNA序列	http：//grna.ctegd.uga.edu
gRNA- seqret	DNA 序列；基因名称	https：//grna.jgi.doe.gov
CRISPOR	DNA 序列；种属；PAM	http：//crispor.tefor.net/
DeepCas13	sgRNA；目标RNA的DNA序列	http：//deepcas13.weililab.org/

工具 Tool	输入 Input	网址 Web site
CHOPCHOP	DNA 序列；基因名称；基因组位置	https：//chopchop.cbu.uib.no
CRISPRDB	基因名称	https：//crisprdb.org
CRISPRscan	DNA 序列	https：//www.crisprscan.org
Synthego	基因名称	https：//design.synthego.com
EuPaGDT	DNA序列	http：//grna.ctegd.uga.edu
gRNA- seqret	DNA 序列；基因名称	https：//grna.jgi.doe.gov
CRISPOR	DNA 序列；种属；PAM	http：//crispor.tefor.net/
DeepCas13	sgRNA；目标RNA的DNA序列	http：//deepcas13.weililab.org/

CRISPR类型 CRISPR type	靶标类型 Target	gRNA设计相同点 Similarity in gRNA design	gRNA设计不同点 Difference in gRNA design
Cas9	miRNA基因	1. 有PAM要求	Drosha、Dicer加工位点及种子序列所对应的基因序列
	circRNA基因	2. 种子序列区无错配	靶向环状外显子或基因座两侧的内含子互补序列
	lncRNA基因	3. 间隔序列GC含量高于50%	pgRNA；靶向剪接位点；BESST策略
Cas13	miRNA	1. 部分有PFS要求	pri/pre-miRNA上的Drosha、Dicer加工位点及种子序列
	circRNA	2. gRNA长度为23-30 bp较佳	circRNA的BSJ位点
	lncRNA	3. 间隔序列GC含量高于50%	gRNA集合；靶向RNA单链区

CRISPR类型 CRISPR type	靶标类型 Target	gRNA设计相同点 Similarity in gRNA design	gRNA设计不同点 Difference in gRNA design
Cas9	miRNA基因	1. 有PAM要求	Drosha、Dicer加工位点及种子序列所对应的基因序列
	circRNA基因	2. 种子序列区无错配	靶向环状外显子或基因座两侧的内含子互补序列
	lncRNA基因	3. 间隔序列GC含量高于50%	pgRNA；靶向剪接位点；BESST策略
Cas13	miRNA	1. 部分有PFS要求	pri/pre-miRNA上的Drosha、Dicer加工位点及种子序列
	circRNA	2. gRNA长度为23-30 bp较佳	circRNA的BSJ位点
	lncRNA	3. 间隔序列GC含量高于50%	gRNA集合；靶向RNA单链区

[1]	崔海洋, 谭淼, 全壮, 陈红利, 董艳敏, 唐立春. 利用Cas9TX实现非病毒TRAC定点整合制备T细胞[J]. 生物技术通报, 2024, 40(9): 190-197.
[2]	侯文婷, 孙琳, 张艳军, 董合忠. 基因编辑技术在棉花种质创新和遗传改良中的应用[J]. 生物技术通报, 2024, 40(7): 68-77.
[3]	陈墨岩, 祝诚. 基于CRISPR/Cas12a的生物传感平台的机制研究及应用[J]. 生物技术通报, 2024, 40(7): 90-98.
[4]	朱恬仪, 孔桂美, 焦红梅, 郭停停, 乌日汗, 刘翠翠, 高成凤, 李国才. CRISPR/Cas9介导的adeG基因敲除大肠杆菌细菌模型的建立[J]. 生物技术通报, 2024, 40(2): 55-64.
[5]	高登科, 马白荣, 郭怡莹, 刘薇, 刘田, 靳亚平, 江舟, 陈华涛. 利用CRISPR/Cas9技术构建Quaking敲除的小鼠胚胎成纤维细胞株[J]. 生物技术通报, 2024, 40(2): 65-72.
[6]	张宏民, 龙雯, 劳筱清, 陈雯妍, 商雪梅, 王洪连, 王丽, 粟宏伟, 沈宏萍, 沈宏春. 利用CRISPR/Cas9技术构建Pmepa1基因敲除的TCMK1小鼠肾小管上皮细胞系[J]. 生物技术通报, 2024, 40(2): 73-79.
[7]	刘佳慧, 刘叶, 花尔并, 王猛. 谷氨酸棒杆菌中胞嘧啶碱基编辑工具的PAM拓展[J]. 生物技术通报, 2023, 39(9): 49-57.
[8]	陈小玲, 廖东庆, 黄尚飞, 陈英, 芦志龙, 陈东. 利用CRISPR/Cas9系统改造酿酒酵母的研究进展[J]. 生物技术通报, 2023, 39(8): 148-158.
[9]	杨玉梅, 张坤晓. 应用CRISPR/Cas9技术建立ERK激酶相分离荧光探针定点整合的稳定细胞株[J]. 生物技术通报, 2023, 39(8): 159-164.
[10]	施炜涛, 姚春鹏, 魏文康, 王蕾, 房元杰, 仝钰洁, 马晓姣, 蒋文, 张晓爱, 邵伟. 利用CRISPR/Cas9技术构建MDH2敲除细胞株及抗呕吐毒素效应研究[J]. 生物技术通报, 2023, 39(7): 307-315.
[11]	刘晓燕, 祝振亮, 史广宇, 华梓宇, 杨晨, 张涌, 刘军. 乳腺生物反应器的表达优化策略[J]. 生物技术通报, 2023, 39(5): 77-91.
[12]	吕宇婧, 吴丹丹, 孔春艳, 杨宇, 龚明. 小桐子XTH基因家族和与之互作的miRNAs的全基因组鉴定及其在低温适应中的作用[J]. 生物技术通报, 2023, 39(2): 147-160.
[13]	程静雯, 曹磊, 张艳敏, 叶倩, 陈敏, 谭文松, 赵亮. CHO细胞多基因工程改造策略的建立及应用[J]. 生物技术通报, 2023, 39(2): 283-291.
[14]	黄文莉, 李香香, 周炆婷, 罗莎, 姚维嘉, 马杰, 张芬, 沈钰森, 顾宏辉, 王建升, 孙勃. 利用CRISPR/Cas9技术靶向编辑青花菜BoZDS[J]. 生物技术通报, 2023, 39(2): 80-87.
[15]	王兵, 赵会纳, 余婧, 陈杰, 骆梅, 雷波. 利用CRISPR/Cas9系统研究REVOLUTA参与烟草叶芽发育的调控[J]. 生物技术通报, 2023, 39(10): 197-208.