CRISPR-Cas基因编辑系统升级：聚焦Cas蛋白和PAM
收稿日期: 2019-11-12
修回日期: 2020-02-26
网络出版日期: 2020-03-04
基金资助
国家自然科学基金项目资助编号(81201181)
Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM
Received date: 2019-11-12
Revised date: 2020-02-26
Online published: 2020-03-04
Supported by
Supported by the National Natural Science Foundation of China No(81201181)
以CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins)系统为代表的基因编辑技术的出现极大地促进了人类改造自然界物种的能力。在医疗、工业、农业等多个研究领域,基因编辑技术正在被广泛应用。Cas蛋白是CRISPR-Cas系统的功能蛋白,不同类型的Cas蛋白在其自身活性、识别位点、切割末端、RNA需求等方面具有不同的特性。PAM (protospacer adjacent motif)是靶位点附近的若干个碱基,对Cas蛋白识别靶序列至关重要,也是CRISPR-Cas系统发挥功效的关键特性之一。目前已有多种不同的PAM鉴定方法被报道。本文对Cas蛋白的寻找、Cas蛋白突变体筛选及PAM的确定方法(含PAM谱拓展)进行了综述,以期为新型基因编辑工具的发展和优化提供借鉴。
关键词： 基因编辑; CRISPR-Cas系统; Cas蛋白; PAM; 定向进化
唐连超, 谷峰 . CRISPR-Cas基因编辑系统升级：聚焦Cas蛋白和PAM[J]. 遗传, 2020 , 42(3) : 236 -249 . DOI: 10.16288/j.yczz.19-297
The emergence of the gene editing technology, especial CRISPR-Cas (clustered regularly intersected short palindromic repeats and CRISPR associated proteins), has greatly promoted the ability of human beings to transform natural species. It has been widely harnessed for the engineering in the medical, industrial, agricultural and other fields. The key component of the CRISPR-Cas system, the Cas protein, possesses its specific features, including self-activity, recognition site, cutting end and guide RNA. PAM (protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we summarize the searching for the Cas protein, the identification of Cas mutants with desired traits and the mapping of the PAM (including the extending of PAM spectrum), in order to provide a reference for the development and optimization of next-generation gene editing tools.
Key words： gene editing; CRISPR-Cas system; Cas protein; PAM; directed evolution
| [1] | Bak RO, Gomez-Ospina N, Porteus MH . Gene editing on center stage. Trends Genet, 2018,34(8):600-611. |
| [2] | Zhang F, Wen Y, Guo X . CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet, 2014,23(R1):R40-R46. |
| [3] | Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 2016, 353(6299): aaf5573. |
| [4] | Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F . Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 2017,356(6336):438-442. |
| [5] | Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F . Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science, 2018,360(6387):439-444. |
| [6] | Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F . Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, 2015,517(7536):583-588. |
| [7] | Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR . Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016,533(7603):420-424. |
| [8] | Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 2016, 353(6305): aaf8729. |
| [9] | Zong Y, Gao CX . Progress on base editing systems. Hereditas(Beijing), 2019,41(9):777-800. |
| [9] | 宗媛, 高彩霞 . 碱基编辑系统研究进展. 遗传, 2019,41(9):777-800. |
| [10] | Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR . Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 2019,576(7785):149-157. |
| [11] | Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P . CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007,315(5819):1709-1712. |
| [12] | Doudna JA, Charpentier E . Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 2014,346(6213):1258096. |
| [13] | Hsu PD, Lander ES, Zhang F . Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014,157(6):1262-1278. |
| [14] | Van Der Oost J, Westra ER, Jackson RN, Wiedenheft B . Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat Rev Microbiol, 2014,12(7):479-492. |
| [15] | Barrangou R, Doudna JA . Applications of CRISPR technologies in research and beyond. Nat Biotechnol, 2016,34(9):933-941. |
| [16] | Jansen R, Embden JD, Gaastra W, Schouls LM . Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol, 2002,43(6):1565-1575. |
| [17] | Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E . Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol, 2005,60(2):174-182. |
| [18] | Shah SA, Erdmann S, Mojica FJ, Garrett RA . Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol, 2013,10(5):891-899. |
| [19] | Westra ER, Semenova E, Datsenko KA, Jackson RN, Wiedenheft B, Severinov K, Brouns SJ . Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition. PLoS Genet, 2013,9(9):e1003742. |
| [20] | Marraffini LA, Sontheimer EJ . Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature, 2010,463(7280):568-571. |
| [21] | Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337(6096):816-821. |
| [22] | Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE, Church GM . RNA-guided human genome engineering via Cas9. Science, 2013,339(6121):823-826. |
| [23] | Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, Hsu PD, Wu XB, Jiang WY, Marraffini LA, Zhang F . Multiplex genome engineering using CRISPR/Cas systems. Science, 2013,339(6121):819-823. |
| [24] | Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F . Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013,8(11):2281-2308. |
| [25] | Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F . In vivo genome editing using Staphylococcus aureus Cas9. Nature, 2015,520(7546):186-191. |
| [26] | Gu F . The new “dark horse” of genome editing tool: FnCpf1 has effective genome editing activity in human cells. Hereditas(Beijing), 2017,39(10):947-949. |
| [26] | 谷峰 . 基因组编辑工具新晋“黑马”:FnCpf1在人类细胞内具备有效基因组编辑活性. 遗传, 2017,39(10):947-949. |
| [27] | He XB, Gu F . Genome-editing: focus on the off-target effects. Chin J Biotechnol, 2017,33(10):1757-1775. |
| [27] | 何秀斌, 谷峰 . 基因组编辑脱靶研究进展. 生物工程学报, 2017,33(10):1757-1775. |
| [28] | Schunder E, Rydzewski K, Grunow R, Heuner K . First indication for a functional CRISPR/Cas system in Francisella tularensis. Int J Med Microbiol, 2013,303(2):51-60. |
| [29] | Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, Van Der Oost J, Regev A, Koonin EV, Zhang F,. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015,163(3):759-771. |
| [30] | Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F, Koonin EV . Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell, 2015,60(3):385-397. |
| [31] | Burstein D, Harrington LB, Strutt SC, Probst AJ, Anantharaman K, Thomas BC, Doudna JA, Banfield JF . New CRISPR-Cas systems from uncultivated microbes. Nature, 2016,542(7640):237-241. |
| [32] | Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Br?tzmann J, Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA . CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature, 2019,566(7743):218-223. |
| [33] | Gao L, Cox D, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F . Engineered Cpf1 variants with altered PAM specificities. Nate Biotechnol, 2017,35(8):789-792. |
| [34] | Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JR, Aryee MJ, Joung JK . Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 2015,523(7561):481-485. |
| [35] | Chen ZL, Zhao HM . A highly sensitive selection method for directed evolution of homing endonucleases. Nucleic Acids Res, 2005,33(18):e154. |
| [36] | Esvelt KM, Carlson JC, Liu DR . A system for the continuous directed evolution of biomolecules. Nature, 2011,472(7344):499-503. |
| [37] | Hu JH, Miller SM, Geurts MH, Tang WX, Chen LW, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR . Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 2018,556(7699):57-63. |
| [38] | Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, Noda T, Abudayyeh OO, Gootenberg JS, Mori H, Oura S, Holmes B, Tanaka M, Seki M, Hirano H, Aburatani H, Ishitani R, Ikawa M, Yachie N, Zhang F, Nureki O . Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science, 2018,361(6408):1259-1262. |
| [39] | He X, Wang Y, Yang F, Wang B, Xie H, Gu L, Zhao T, Liu X, Zhang D, Ren Q, Liu X, Liu Y, Gao C, Gu F . Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNA Gln-processing system in human cells . J Biol Chem, 2019,294(23):9308-9315. |
| [40] | Horvath P, Romero DA, Coute-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R . Diversity, activity, and evolution of CRISPR loci in streptococcus thermophilus. J Bacteriol, 2008,190(4):1401-1412. |
| [41] | Deveau H, Barrangou R, Garneau JE, Labonte J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S . Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol, 2008,190(4):1390-1400. |
| [42] | Mojica FJ, Diez-Villasenor C, García-Martinez J, Almendros C . Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology, 2009,155(Pt 3):733-740. |
| [43] | Wiedenheft B, Van DE, Bultema JB, Waghmare SP, Zhou K, Barendregt A, Westphal W, Heck AJ, Boekema EJ, Dickman MJ . RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci USA, 2011,108(25):10092-10097. |
| [44] | Mulepati S, Héroux A, Bailey S . Structural biology. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science, 2014,345(6203):1479-1484. |
| [45] | Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, Kaplan M, Iavarone AT, Charpentier E, Nogales E, Doudna JA . Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 2014,343(6176):1247997. |
| [46] | Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O . Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 2014,156(5):935-949. |
| [47] | Westra ER, Van Erp PB, Kunne T, Wong SP, Staals RH, Seegers CL, Bollen S, Jore MM, Semenova E, Severinov K, De Vos WM, Dame RT, De Vries R, Brouns SJ, Van Der Oost J. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell, 2012,46(5):595-605. |
| [48] | Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, Van Der Oost J, Brouns SJ, Severinov K. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci USA, 2011,108(25):10098-10103. |
| [49] | Elmore J, Deighan T, Westpheling J, Terns RM, Terns MP . DNA targeting by the type I-G and type I-A CRISPR-Cas systems of Pyrococcus furiosus. Nucleic Acids Res, 2015,43(21):10353-10363. |
| [50] | Biswas A, Gagnon JN, Brouns SJ, Fineran PC, Brown CM . CRISPRTarget: bioinformatic prediction and analysis of crRNA targets. RNA Biol, 2013,10(5):817-827. |
| [51] | Xue C, Seetharam AS, Musharova O, Severinov K, Brouns S JJ, Severin AJ, Sashital DG . CRISPR interference and priming varies with individual spacer sequences. Nucleic Acids Res, 2015,43(22):10831-10847. |
| [52] | Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR . High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol, 2013,31(9):839-843. |
| [53] | Karvelis T, Gasiunas G, Young J, Bigelyte G, Silanskas A, Cigan M, Siksnys V . Rapid characterization of CRISPR- Cas9 protospacer adjacent motif sequence elements. Genome Biol, 2015,16:253. |
| [54] | Leenay RT, Maksimchuk KR, Slotkowski RA, Agrawal RN, Gomaa AA, Briner AE, Barrangou R, Beisel CL . Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol Cell, 2016,62(1):137-147. |
| [55] | Tang LC, Yang FY, He XX, Xie HH, Liu XY, Fu JH, Xi HT, Lu XS, Liu CB, Song ZM, Qu J, Zhao JZ, Gu F . Efficient cleavage resolves PAM preferences of CRISPR- Cas in human cells. Cell Regen, 2019,8(2):44-50. |
| [56] | Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F . DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 2013,31(9):827-832. |
| [57] | Zhang YL, Ge XL, Yang FY, Zhang LP, Zheng JR, Tan XF, Jin ZB, Qu J, Gu F . Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci Rep, 2014,4:5405. |
| [58] | Tu MJ, Lin L, Cheng YL, He XB, Sun HH, Xie HH, Fu JH, Liu CB, Li J, Chen D, Xi HT, Xue DY, Liu Q, Zhao JZ, Gao CX, Song ZM, Qu J, Gu F . A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res, 2017,45(19):11295-11304. |
| [59] | Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS . A CRISPR/CAS system mediates bacterial innate immune evasion and virulence. Nature, 2013,497(7448):254-257. |
| [60] | Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS, Schoen C, Vogel J, Sontheimer EJ . Processing- independent CRISPR RNAs limit natural transformation in neisseria meningitidis. Molecular Cell, 2013,50(4):488-503. |
| [61] | Chylinski K, Le Rhun A, Charpentier E . The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol, 2013,10(5):726-737. |
| [62] | Hirano H, Gootenberg JS, Horii T, Abudayyeh OO, Kimura M, Hsu PD, Nakane T, Ishitani R, Hatada I, Zhang F, Nishimasu H, Nureki O . Structure and engineering of francisella novicida Cas9. Cell, 2016,164(5):950-961. |
| [63] | Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA . RNA- guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 2013,31(3):233-239. |
| [64] | Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM . Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 2013,10(11):1116-11121. |
| [65] | Yamada M, Watanabe Y, Gootenberg JS, Hirano H, Ran FA, Nakane T, Ishitani R, Zhang F, Nishimasu H, Nureki O. Crystal Structure of the minimal Cas9 from campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 systems. Mol Cell, 2017, 65(6): 1109-1121.e3. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
