Design and Application of Guide RNAs for Non-coding RNAs

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

Abstract

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

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.

Figures/Tables 4

References 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.

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]	CUI Hai-yang, TAN Miao, QUAN Zhuang, CHEN Hong-li, DONG Yan-min, TANG Li-chun. Generation of Virus-free TRAC-knocked-in T Cells Using Cas9TX [J]. Biotechnology Bulletin, 2024, 40(9): 190-197.
[2]	HOU Wen-ting, SUN Lin, ZHANG Yan-jun, DONG He-zhong. Application of Gene-editing Technology for Germplasm Innovation and Genetic Improvement in Cotton [J]. Biotechnology Bulletin, 2024, 40(7): 68-77.
[3]	CHEN Mo-yan, ZHU Cheng. Mechanism Study and Application of CRISPR/Cas12a-based Biosensing Platform [J]. Biotechnology Bulletin, 2024, 40(7): 90-98.
[4]	ZHU Tian-yi, KONG Gui-mei, JIAO Hong-mei, GUO Ting-ting, WU Ri-han, LIU Cui-cui, GAO Cheng-feng, LI Guo-cai. Establishment of A Bacterial Model of CRISPR/Cas9 Mediated adeG Gene Knockout in Escherichia coli [J]. Biotechnology Bulletin, 2024, 40(2): 55-64.
[5]	GAO Deng-ke, MA Bai-rong, GUO Yi-ying, LIU Wei, LIU Tian, JIN Ya-ping, JIANG Zhou, CHEN Hua-tao. Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology [J]. Biotechnology Bulletin, 2024, 40(2): 65-72.
[6]	ZHANG Hong-min, LONG Wen, LAO Xiao-qing, CHEN Wen-yan, SHANG Xue-mei, WANG Hong-lian, WANG Li, SU Hong-wei, SHEN Hong-ping, SHEN Hong-chun. Construction of Pmepa1 Knockout TCMK1 Mouse Renal Tubular Epithelial Cell Line Using CRISPR/Cas9 Technology [J]. Biotechnology Bulletin, 2024, 40(2): 73-79.
[7]	LIU Jia-hui, LIU Ye, HUA Er-bing, WANG Meng. PAM Extension of Cytosine Base Editing Tool in Corynebacterium glutamicum [J]. Biotechnology Bulletin, 2023, 39(9): 49-57.
[8]	CHEN Xiao-ling, LIAO Dong-qing, HUANG Shang-fei, CHEN Ying, LU Zhi-long, CHEN Dong. Advances in CRISPR/Cas9 System Modifying Saccharomycescerevisiae [J]. Biotechnology Bulletin, 2023, 39(8): 148-158.
[9]	YANG Yu-mei, ZHANG Kun-xiao. Establishing a Stable Cell Line with Site-specific Integration of ERK Kinase Phase-separated Fluorescent Probe Using CRISPR/Cas9 Technology [J]. Biotechnology Bulletin, 2023, 39(8): 159-164.
[10]	SHI Wei-tao, YAO Chun-peng, WEI Wen-Kang, WANG Lei, FANG Yuan-jie, TONG Yu-jie, MA Xiao-jiao, JIANG Wen, ZHANG Xiao-ai, SHAO Wei. Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect [J]. Biotechnology Bulletin, 2023, 39(7): 307-315.
[11]	LIU Xiao-yan, ZHU Zhen-liang, SHI Guang-yu, HUA Zi-yu, YANG Chen, ZHANG Yong, LIU Jun. Strategies to Optimize the Expression of Mammary Gland Bioreactor [J]. Biotechnology Bulletin, 2023, 39(5): 77-91.
[12]	LV Yu-jing, WU Dan-dan, KONG Chun-yan, YANG Yu, GONG Ming. Genome-wide Identification of XTH Gene Family and Their Interacting miRNAs and Possible Roles in Low Temperature Adaptation in Jatropha curcas L. [J]. Biotechnology Bulletin, 2023, 39(2): 147-160.
[13]	CHENG Jing-wen, CAO Lei, ZHANG Yan-min, YE Qian, CHEN Min, TAN Wen-song, ZHAO Liang. Establishment and Application of Multigene Engineering Transformation Strategy for CHO Cells [J]. Biotechnology Bulletin, 2023, 39(2): 283-291.
[14]	HUANG Wen-li, LI Xiang-xiang, ZHOU Wen-ting, LUO Sha, YAO Wei-jia, MA Jie, ZHANG Fen, SHEN Yu-sen, GU Hong-hui, WANG Jian-sheng, SUN Bo. Targeted Editing of BoZDS in Broccoli by CRISPR/Cas9 Technology [J]. Biotechnology Bulletin, 2023, 39(2): 80-87.
[15]	WANG Bing, ZHAO Hui-na, YU Jing, CHEN Jie, LUO Mei, LEI Bo. Regulation of Leaf Bud by REVOLUTA in Tobacco Based on CRISPR/Cas9 System [J]. Biotechnology Bulletin, 2023, 39(10): 197-208.