嗜冷黄杆菌CRISPR/Cas系统生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.06.005

Abstract

Abstract: To develop a feasible genome editing technology based on CRISPR/Cas system of Flavobacterium psychrophilum,the CRISPR/Cas system structure of Flavobacterium psychrophilum and its mechanism of action were analyzed by bioinformatics.The complete genome sequences of eight Flavobacterium psychrophilum strains were available in the GenBank database,and CRISPRCasFinder software was used to identify both clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated (Cas) proteins in the Flavobacterium psychrophilum genomes.The repeats and spacers of the CRISPR arrays were further identified using CRISPRFinder software,and the nucleotide sequence identities of cas genes were analyzed using Mega X software.Nucleotide alignment was performed with repeat sequence to search for the antirepeat portion of the trans-activating CRISPR RNA (tracrRNA),and the transcription terminator of the tracrRNA was predicted using ARNold software.The promoters of tracrRNA and crRNA precursor (pre-crRNA) were predicted using BPROM software.The nucleotide sequence identities of spacers were analyzed using Clustal X software,and CRISPRTarget software was used to predict protospacers matching the unique spacers and their protospacer adjacent motifs (PAMs).The PAM of the matching protospacers was obtained using WebLogo software.The results showed that all 8 strains had only full intact a CRISPR/Cas9 system,including a CRISPR array and three Cas proteins.The CRISPR array consisted of short repeated sequences (repeats) interspersed with short variable sequences (spacers).Spacers in all eight CRISPR arrays were 46 bp long,and the nucleotide sequence of repeat were highly similar.Spacers had a size between 29 and 31 bp,and the numbers of spacers varied from 20 to 41.All 8 CRISPR/Cas9 systems included a set of only three Cas proteins (Cas9,Cas1 and Cas2) which were highly conserved.The tracrRNA sequences shared 100% identity at the nucleotide level and found upstream of the cas9 gene.There was a stretch of 24 nucleotides where 23 nucleotides of the tracrRNA matched the repeat sequence.Each repeat carried its own minimal promoter and the pre-crRNA transcription initiated independently within each spacer.Spacer alignments among 8 different strains revealed that newly acquired spacers were integrated both at the 5'end of the CRISPR array and internally.13 of the 65 unique spacers could be mapped as protospacers on phages or plasmids.Protospacer alignments revealed an apparent PAM of 5'-GANTTTT-3'recognized by the Cas9.Taken together,the CRISPR/Cas9 system could be used to develop a feasible genome editing technology suitable for Flavobacterium psychrophilum in theory.

Key words: Flavobacterium psychrophilum; CRISPR/Cas9 system; Cas proteins; tracrRNA; repeats; spacers

CLC Number:

S941.42

CHEN Fuguang, LU Tongyan, LI Shaowu. Bioinformatic Analysis of CRISPR/Cas System in Flavobacterium psychrophilum[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 1927-1938.

References

[1] HAMPTON H G,WATSON B N J,FINERAN P C.The arms race between bacteria and their phage foes[J].Nature,2020,577(7790):327-336.
[2] 胡丽,陈实.细菌CRISPR-Cas系统的研究进展[J].微生物学报,2017,57(11):1643-1652. HU L,CHEN S.Research progress of CRISPR-Cas system in bacteria[J].Acta Microbiologica Sinica,2017,57(11):1643-1652.(in Chinese)
[3] 向华.来自细菌的魔剪:2020年诺贝尔化学奖[J].微生物学通报,2020,47(11):3491-3493. XIANG H.Genetic scissors from bacteria:The 2020 Nobel prize in chemistry[J].Microbiology China,2020,47(11):3491-3493.(in Chinese)
[4] KOONIN E V,MAKAROVA K S,ZHANG F.Diversity,classification and evolution of CRISPR-Cas systems[J].Current Opinion in Microbiology,2017,37:67-78.
[5] 杨兰.Lactobacillus paracasei CRISPR-Cas系统多样性分析及PAM验证[D].北京:中国农业科学院,2020. YANG L.Diversity analysis and PAM verification of Lactobacillus paracasei CRISPR-Cas system[D].Beijing:Chinese Academy of Agricultural Scicences,2020.(in Chinese)
[6] SHMAKOV S,SMARGON A,SCOTT D,et al.Diversity and evolution of class 2 CRISPR-Cas systems[J].Nature Reviews Microbiology,2017,15(3):169-182.
[7] ZHANG Y,HEIDRICH N,AMPATTU B J,et al.Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis[J].Molcular Cell,2013,50(4):488-503.
[8] ZHANG Y,RAJAN R,SEIFERT H S,et al.DNase H activity of Neisseria meningitidis cas9[J].Molecular Cell,2015,60(2):242-255.
[9] ZHU Y,GAO A,ZHAN Q,et al.Diverse mechanisms of CRISPR-Cas9 inhibition by type ⅡC anti-CRISPR proteins[J].Molecular Cell,2019,74(2):296-309.
[10] KIM E,KOO T,PARK S W,et al.In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni[J].Nature Communications,2017,8:14500.
[11] EDRAKI A,MIR A,IBRAHEIM R,et al.A compact,high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing[J].Molecular Cell,2019,73(4):714-726.
[12] 柴静茹,王荻,卢彤岩,等.嗜冷黄杆菌及细菌性冷水病的研究进展[J].大连海洋大学学报,2020,35(5):755-761. CHAI J R,WANG D,LU T Y,et al.Research progress on Flavobacterium psychrophilum and bacterial coldwater disease:A review[J].Journal of Dalian Ocean University,2020,35(5):755-761.(in Chinese)
[13] BARBIER P,ROCHAT T,MOHAMMED H H,et al.The type IX secretion system is required for virulence of the fish pathogen Flavobacterium psychrophilum[J].Applied and Environmental Microbiology,2020,86(16):e00799-20.
[14] JIANG W,BIKARD D,COX D,et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J].Nature Biotechnology,2013,31(3):233-239.
[15] VENTO J M,CROOK N,BEISEL C L.Barriers to genome editing with CRISPR in bacteria[J].Journal of Industrial Microbiology & Biotechnology,2019,46(9-10):1327-1341.
[16] ALVAREZ B,SECADES P,MCBRIDE M J,et al.Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum[J].Applied and Environmental Microbiology,2004,70(1):581-587.
[17] COUVIN D,BERNHEIM A,TOFFANO-NIOCHE C,et al.CRISPRCasFinder,an update of CRISRFinder,includes a portable version,enhanced performance and integrates search for Cas proteins[J].Nucleic Acids Research,2018,46(W1):W246-W251.
[18] GRISSA I,VERGNAUD G,POURCEL C.CRISPRFinder:A web tool to identify clustered regularly interspaced short palindromic repeats[J].Nucleic Acids Research,2007,35:W52-W57.
[19] KUMAR S,STECHER G,LI M,et al.MEGA X:Molecular evolutionary genetics analysis across computing platforms[J].Molecular Biology and Evolution,2018,35(6):1547-1549.
[20] FAURE G,SHMAKOV S A,MAKAROVA K S,et al.Comparative genomics and evolution of trans-activating RNAs in class 2 CRISPR-Cas systems[J].RNA Biology,2019,16(4):435-448.
[21] BRINER A E,HENRIKSEN E D,BARRANGOU R.Prediction and validation of native and engineered Cas9 guide sequences[J].Cold Spring Harbor Protocols,2016,2016(7):628-634.
[22] NAVILLE M,GHUILLOT-GAUDEFFROY A,MARCHAIS A,et al.ARNold:A web tool for the prediction of Rho-independent transcription terminators[J].RNA Biology,2011,8(1):11-13.
[23] LARKIN M A,BLACKSHIELDS G,BROWN N P,et al.Clustal W and Clustal X version 2.0[J].Bioinformatics,2007,23(21):2947-2948.
[24] BISWAS A,GAGNON J N,BROUNS S J,et al.CRISPRTarget:Bioinformatic prediction and analysis of crRNA targets[J].RNA Biology,2013,10(5):817-827.
[25] CROOKS G E,HON G,CHANDONIA J M,et al.WebLogo:A sequence logo generator[J].Genome Research,2004,14(6):1188-1190.
[26] GOMEZ E,ALVAREZ B,DUCHAUD E,et al.Development of a markerless deletion system for the fish-pathogenic bacterium Flavobacterium psychro-philum[J].PLoS One,2015,10(2):e0117969.
[27] PENEWIT K,HOLMES E A,MCLEAN K,et al.Efficient and scalable precision genome editing in Staphylococcus aureus through conditional recombineering and CRISPR/Cas9-mediated counterselection[J].mBio,2018,9(1):e00067-18.
[28] HELER R,SAMAI P,MODELL J W,et al.Cas9 specifies functional viral targets during CRISPR-Cas adaptation[J].Nature,2015,519(7542):199-202.
[29] KA D,JANG D M,HAN B W,et al.Molecular organization of the type Ⅱ-A CRISPR adaptation module and its interaction with Cas9 via Csn2[J].Nucleic Acids Research,2018,46(18):9805-9815.
[30] HOOTON S P,CONNERTON I F.Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harboring a CRISPR-like Cas4 protein[J].Frontiers in Microbiology,2015,5:744.
[31] MOSTERD C,MOINEAU S.Characterization of a type Ⅱ-A CRISPR-Cas system in Streptococcus mutans[J].mSphere,2020,5(3):e00235-20.

Bioinformatic Analysis of CRISPR/Cas System in Flavobacterium psychrophilum

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 9

Recommended Articles

Metrics

Comments

[1]	MA Jing, ZHANG Yanyan, JIA Xinyue, MA Xun, WANG Zhengrong, BO Xinwen. Research Progress of CRISPR/Cas9 System in Parasite Genome Editing [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3713-3725.
[2]	HUANG Huilan, LI Wenjun, XIE Zimin, YANG Huihu, CUI Huiyi, HUANG Shujian, ZHANG Xuelian. Molecular Cloning and Expression of Duck IFIT5 Gene and Preparation of Its Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3713-3721.
[3]	ZHANG Lei, ZHANG Haibo, ZHANG Jingqi, ZHANG Yang, CHANG Guobin, CHEN Guohong, WANG Jian. Research Progress on Application of CRISPR/Cas9 System in Poultry [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 140-147.
[4]	QIU Guiru, WANG Zheng, HE Tingting, GENG Zhaoyu, JIN Sihua. Research Advances on CRISPR/Cas9 System and Its Application in Genome Editing in Chickens [J]. , 2018, 45(6): 1590-1598.
[5]	REN Hong-yan, Xiao Hong-wei, LI Xiao-min, XU Zhen-yu, BI Yan-zhen, HUA Zai-dong, ZHENG Xin-min. Establishment and Application of Nested PCR Method for Detecting Mutation in Single Cell [J]. , 2016, 43(10): 2566-2571.
[6]	LI Cong, CAO Wen-guang. Knockout of Myostatin Gene by CRISPR/Cas9 System in Ovine Fetal Fibroblasts [J]. , 2015, 42(11): 2813-2821.
[7]	LI Xiao-jun, CHEN Yu, TANG Xing-zhong, HONG Qing-lin. PCR Amplification and Sequence Analysis of ITS rDNA of Anisakid larvae in Imported Pacific Cod [J]. , 2012, 39(6): 90-92.
[8]	HE Xian-hui;CHEN WU;HE Le-tian;WANG Pei-yuan;YUAN Zi-guo;LIN Rui-qing. PCR Amplification and Sequence Analysis of Internal Transcribed Spacers(ITS) of Ascarids from Puma concolor and Ursus thibetanus [J]. , 2010, 37(8): 41-44.
[9]	HU Jindong;WU Yuanzheng;YANG Hetong;. Cloning of Gene PR-39 from Pig and Construction of PR-39 as Tandem Repeats Expression Vector in E coli [J]. , 2008, 1(12): 56-59.