基于体外组装核糖核蛋白形式的CRISPR/Cas9基因编辑方法研究进展
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摘要
CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated)系统是近年来发展起来的新型的基因编辑技术,在生物医学领域得到广泛应用。CRISPR/Cas9系统需要在gRNA存在的条件下通过Cas9蛋白实现对基因组的定点编辑,通常情况下以慢病毒感染或质粒转染等方式提供Cas9和gRNA。但是,这些方式容易引起免疫反应及基因片段不可控插入,存在一定的风险,限制了CRISPR/Cas9技术在机体的应用。近年来发展起来的基于体外组装的核糖核蛋白(ribonucleoprotein,RNP)转导入胞的策略由于快捷安全、编辑的脱靶率低等优势引起广泛关注。对Cas9 RNP的转导方式及其应用进行了总结,并就其目前存在的问题进行探讨,以期为CRISPR/Cas9技术的进一步发展提供依据,为拓展其应用奠定基础。
CRISPR( clustered ordered interspaced short palindromic repeats)/Cas( CRISPR-associated)system is a novel gene editing technology developed in recent years,which is widely used in the biomedical fields. The Cas9 protein and gRNA is essential for the site-specific editing of the genome by CRISPR/Cas9 system. Usually,Cas9 and gRNA are provided by lentivirus infection or plasmid transfection. However,these approaches are likely to cause adverse effects such as immune reactions and uncontrolled insertion of gene fragments,and limits the application of CRISPR/Cas9 systems. The strategy of RNP transduction based on in vitro assembly of Cas9 and gRNA developed in recent years has attracted wide attention because of its advantages such as rapid,safe and low off-target effect. The way of transduction of Cas9 RNP and its application are summarized,and its current problems are discussed in order to provide basis for the further development of CRISPR/Cas9 technology and lay a foundation for its application.
CRISPR( clustered ordered interspaced short palindromic repeats)/Cas( CRISPR-associated)system is a novel gene editing technology developed in recent years,which is widely used in the biomedical fields. The Cas9 protein and gRNA is essential for the site-specific editing of the genome by CRISPR/Cas9 system. Usually,Cas9 and gRNA are provided by lentivirus infection or plasmid transfection. However,these approaches are likely to cause adverse effects such as immune reactions and uncontrolled insertion of gene fragments,and limits the application of CRISPR/Cas9 systems. The strategy of RNP transduction based on in vitro assembly of Cas9 and gRNA developed in recent years has attracted wide attention because of its advantages such as rapid,safe and low off-target effect. The way of transduction of Cas9 RNP and its application are summarized,and its current problems are discussed in order to provide basis for the further development of CRISPR/Cas9 technology and lay a foundation for its application.
引文
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[29]Li L,He Z Y,Wei X W,et al. Challenges in CRISPR/CAS9delivery:Potential roles of nonviral vectors. Human Gene Therapy,2015,26(7):452-462.
[30]Glass Z,Lee M,Li Y,et al. Engineering the delivery system for CRISPR-based genome editing. Trends in Biotechnology,2018,36(2):173-185.
[31] Kesharwani P,Iyer A K. Recent advances in dendrimer-based nanovectors for tumor-targeted drug and gene delivery. Drug Discovery Today,2015,20(5):536-547.
[32] Jeong S H,Jang J H,Cho H Y,et al. Soft-and hard-lipid nanoparticles:a novel approach to lymphatic drug delivery.Archives of Pharmacal Research,2018,41:797-814.
[33]Diaz D,Care A,Sunna A. Bioengineering strategies for proteinbased nanoparticles. Genes,2018,9(7):370.
[34]Sun W,Ji W,Hall J M,et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing.Angewandte Chemie,2015,54(41):12029-2033.
[35]Mout R,Ray M,Yesilbag T G,et al. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano,2017,11(3):2452-2458.
[36] Lee K,Conboy M,Park H M,et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homologydirected DNA repair. Nature Biomedical Engineering,2017,1(11):889-901.
[37] Wang M,Alberti K,Varone A,et al. Enhanced intracellular siRNA delivery using bioreducible lipid-like nanoparticles.Advanced Healthcare Materials,2014,3(9):1398-1403.
[38]Ramakrishna S,Kwaku D A B,Beloor J,et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Research,2014,24(6):1020-1027.
[39]Jones S W,Christison R,Bundell K,et al. Characterisation of cell-penetrating peptide-mediated peptide delivery. British Journal of Pharmacology,2005,145(8):1093-1102.
[40]Kaestner L,Scholz A,Lipp P. Conceptual and technical aspects of transfection and gene delivery. Bioorganic&Medicinal Chemistry Letters,2015,25(6):1171-1176.
[41] Rouet R,Thuma B A,Roy M D,et al. Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing. Journal of the American Chemical Society,2018,140(21):6596-6603.
[2]Qi L S,Larson M H,Gilbert L A,et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell,2013,152(5):1173-1183.
[3] Jiang W,Bikard D,Cox D,et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology,2013,31(3):233-239.
[4] Nekrasov V, Staskawicz B, Weigel D, et al. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9RNA-guided endonuclease. Nature Biotechnology,2013,31(8):691-693.
[5]Doudna J A,Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science,2014,346(6213):1258096.
[6]Zhang Y,Qin W,Lu X,et al. Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nature Communications,2017,8(1):118.
[7] Suzuki K,Tsunekawa Y,Hernandez-Benitez R,et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature,2016,540(7631):144-149.
[8] Jaganathan D,Ramasamy K,Sellamuthu G,et al. CRISPR for crop improvement:an update review. Frontiers in Plant Science,2018,9. DOI:10. 3389/fpls. 2018. 00985.
[9] Gai T,Gersbach C A,Barbas C F,3RD. ZFN,TALEN,and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology,2013,31(7):397-405.
[10] Chandrasegaran S. Recent advances in the use of ZFN-mediated gene editing for human gene therapy. Cell&Gene Therapy Insights,2017,3(1):33-41.
[11] Moscou M J,Bogdanove A J. A simple cipher governs DNA recognition by TAL effectors. Science,2009,326(5959):1501.
[12] Bi H,Yang B. Gene editing with TALEN and CRISPR/Cas in rice. Progress in Molecular Biology and Translational Science,2017,149:81-98.
[13]Woo J W,Kim J,Kwon S I,et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.Nature Biotechnology,2015,33(11):1162-1164.
[14]Chen X,Goncalves M A. Engineered viruses as genome editing devices. Molecular Therapy:The Journal of the American Society of Gene Therapy,2016,24(3):447-457.
[15] Kim S,Kim D,Cho S W,et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9ribonucleoproteins. Genome Research,2014,24(6):1012-1019.
[16]Nishimasu H,Ran F A,Hsu P D,et al. Crystal structure of Cas9in complex with guide RNA and target DNA. Cell,2014,156(5):935-949.
[17] Jinek M,Jiang F,Taylor D W,et al. Structures of Cas9endonucleases reveal RNA-mediated conformational activation.Science,2014,343(6176):1247997.
[18]Bueger A,Lindsay H,Felker A,et al. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.Development,2016,143(11):2025-2037.
[19]Wang H X,Li M,Lee C M,et al. CRISPR/Cas9-based genome editing for disease modeling and therapy:challenges and opportunities for nonviral delivery. Chemical Reviews,2017,117(15):9874-9906.
[20] Schumann K,Lin S,Boyer E,et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proceedings of the National Academy of Sciences of the United States of America,2015,112(33):10437-10442.
[21]Meccariello A,Monti S M,Romanelli A,et al. Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes. Scientific Reports,2017,7(1):10061.
[22]Staahl B T,Benekareddy M,Coulon-Bainier C,et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nature Biotechnology,2017,35(5):431-434.
[23]Lin S,Staahl B T,Alla R K,et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9delivery. e Life,2014,3(6):e04766.
[24]Wang M,Glass Z A,Xu Q. Non-viral delivery of genome-editing nucleases for gene therapy. Gene Therapy,2017,24(3):144-150.
[25]Zuris J A,Thompson D B,Shu Y,et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnology,2015,33(1):73-80.
[26] Liang X,Potter J,Kumar S,et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology,2015,208:44-53.
[27] Kanchiswamy C N,Malnoy M,Velasco R,et al. Non-GMO genetically edited crop plants. Trends in Biotechnology,2015,33(9):489-491.
[28] Yue H,Zhou X,Cheng M,et al. Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing. Nanoscale,2018,10(3):1063-1071.
[29]Li L,He Z Y,Wei X W,et al. Challenges in CRISPR/CAS9delivery:Potential roles of nonviral vectors. Human Gene Therapy,2015,26(7):452-462.
[30]Glass Z,Lee M,Li Y,et al. Engineering the delivery system for CRISPR-based genome editing. Trends in Biotechnology,2018,36(2):173-185.
[31] Kesharwani P,Iyer A K. Recent advances in dendrimer-based nanovectors for tumor-targeted drug and gene delivery. Drug Discovery Today,2015,20(5):536-547.
[32] Jeong S H,Jang J H,Cho H Y,et al. Soft-and hard-lipid nanoparticles:a novel approach to lymphatic drug delivery.Archives of Pharmacal Research,2018,41:797-814.
[33]Diaz D,Care A,Sunna A. Bioengineering strategies for proteinbased nanoparticles. Genes,2018,9(7):370.
[34]Sun W,Ji W,Hall J M,et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing.Angewandte Chemie,2015,54(41):12029-2033.
[35]Mout R,Ray M,Yesilbag T G,et al. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano,2017,11(3):2452-2458.
[36] Lee K,Conboy M,Park H M,et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homologydirected DNA repair. Nature Biomedical Engineering,2017,1(11):889-901.
[37] Wang M,Alberti K,Varone A,et al. Enhanced intracellular siRNA delivery using bioreducible lipid-like nanoparticles.Advanced Healthcare Materials,2014,3(9):1398-1403.
[38]Ramakrishna S,Kwaku D A B,Beloor J,et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Research,2014,24(6):1020-1027.
[39]Jones S W,Christison R,Bundell K,et al. Characterisation of cell-penetrating peptide-mediated peptide delivery. British Journal of Pharmacology,2005,145(8):1093-1102.
[40]Kaestner L,Scholz A,Lipp P. Conceptual and technical aspects of transfection and gene delivery. Bioorganic&Medicinal Chemistry Letters,2015,25(6):1171-1176.
[41] Rouet R,Thuma B A,Roy M D,et al. Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing. Journal of the American Chemical Society,2018,140(21):6596-6603.