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

摘要/Abstract

摘要： 为了开发基于嗜冷黄杆菌CRISPR/Cas系统的基因组编辑技术，本研究对嗜冷黄杆菌的CRISPR/Cas系统结构及其作用机制进行生物信息学分析。从GenBank数据库中获得8株嗜冷黄杆菌的全基因组序列，利用CRISPRCasFinder软件查找成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）结构和CRISPR相关（Cas）蛋白在嗜冷黄杆菌基因组上的数量和分布；利用CRISPRFinder软件分析CRISPR结构的重复序列和间隔序列，并使用Mega X软件对cas基因核苷酸序列做相似性对比；通过与重复序列配对，获得反式激活crRNA （trans-activating CRISPR RNA，tracrRNA）与重复序列配对序列，使用ARNold软件预测tracrRNA基因的终止子；使用BPROM软件预测tracrRNA基因和crRNA前体（pre-CRISPR RNA，pre-crRNA）的启动子；使用Clustal X软件对所有的间隔序列做相似性对比，并使用CRISPRTarget软件对独特的间隔序列配对从而获得原间隔序列（protospacers）及原间隔序列邻近基序（protospacer adjacent motif，PAM）序列；使用WebLogo软件使PAM序列可视化。结果显示，8株嗜冷黄杆菌均含有1个完整的CRISPR/Cas9系统，由1个CRISPR结构和3个Cas蛋白组成。CRISPR结构由短而重复的序列即重复序列和短而可变的序列即间隔序列相间排列组成；重复序列大小为46 bp，核苷酸序列高度保守；间隔序列大小在29~31 bp之间，数量在20~41个之间。Cas蛋白含有Cas9、Cas1和Cas2，并且cas基因核苷酸序列高度保守。8株嗜冷黄杆菌的tracrRNA基因均位于cas9基因上游并且核苷酸序列相似性为100%。tracrRNA上有一段大小为24 bp的核苷酸序列，其中23个核苷酸与重复序列完全配对。每个重复序列均含有一个较短的启动子，可单独启动pre-crRNA的转录。不同菌株的间隔序列比对结果表明，新获得的间隔序列可以插入到嗜冷黄杆菌CRISPR结构的5'端或内部。在65个独特的间隔序列中，13个间隔序列能够配对上原间隔序列，这些原间隔序列均来源于噬菌体或质粒。原间隔序列上游侧翼序列分析结果表明，嗜冷黄杆菌Cas9识别的PAM序列是5‘-GANTTTT-3’。以上结果表明嗜冷黄杆菌的CRISPR/Cas9系统理论上可以开发适用于嗜冷黄杆菌的基因组编辑技术。

关键词: 嗜冷黄杆菌; CRISPR/Cas9系统; Cas蛋白; tracrRNA; 重复序列; 间隔序列

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

中图分类号:

S941.42

陈福广, 卢彤岩, 李绍戊. 嗜冷黄杆菌CRISPR/Cas系统生物信息学分析[J]. 中国畜牧兽医, 2021, 48(6): 1927-1938.

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.

参考文献

[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.