Abstract
Genome-editing technologies have revolutionized the biomedical sciences by providing researchers with the ability to quickly and efficiently modify genes. While programmable nucleases can be introduced into cells using a variety of techniques, their delivery as purified proteins is an effective approach for limiting off-target effects. Here, we describe step-by-step procedures for manufacturing and delivering genome-modifying proteins—including Cas9 ribonucleoproteins (RNPs) and TALE and zinc-finger nucleases—into mammalian cells. Protocols for combining Cas9 RNP with naturally recombinogenic adeno-associated virus (AAV) donor vectors for the seamless insertion of transgenes by homology-directed genome editing are also provided.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161:1169–1175
Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 105:5809–5814
Porteus MH, Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300:763
Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764
Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–651
Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93:1156–1160
Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148
Gersbach CA, Gaj T, Barbas CF 3rd (2014) Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies. Acc Chem Res 47:2309–2318
Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326:1501
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
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 (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573
Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F (2017) RNA targeting with CRISPR-Cas13. Nature 550:280–284
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. Elife 2:e00471
Chu G, Hayakawa H, Berg P (1987) Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15:1311–1326
Hamm A, Krott N, Breibach I, Blindt R, Bosserhoff AK (2002) Efficient transfection method for primary cells. Tissue Eng 8:235–245
Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A 84:7413–7417
Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 92:7297–7301
Roursgaard M, Knudsen KB, Northeved H, Persson M, Christensen T, Kumar PEK, Permin A, Andresen TL, Gjetting T, Lykkesfeldt J, Vesterdal LK, Loft S, Moller P (2016) In vitro toxicity of cationic micelles and liposomes in cultured human hepatocyte (HepG2) and lung epithelial (A549) cell lines. Toxicol In Vitro 36:164–171
Liu J, Shui SL (2016) Delivery methods for site-specific nucleases: achieving the full potential of therapeutic gene editing. J Control Release 244:83–97
Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F (2015) In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33:102–106
Tervo DG, Hwang BY, Viswanathan S, Gaj T, Lavzin M, Ritola KD, Lindo S, Michael S, Kuleshova E, Ojala D, Huang CC, Gerfen CR, Schiller J, Dudman JT, Hantman AW, Looger LL, Schaffer DV, Karpova AY (2016) A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92:372–382
Gaj T, Ojala DS, Ekman FK, Byrne LC, Limsirichai P, Schaffer DV (2017) In vivo genome editing improves motor function and extends survival in a mouse model of ALS. Sci Adv 3:eaar3952
Gaj T, Epstein BE, Schaffer DV (2015) Genome engineering using adeno-associated virus: basic and clinical research applications. Mol Ther 24:58–464
Pruett-Miller SM, Reading DW, Porter SN, Porteus MH (2009) Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels. PLoS Genet 5:e1000376
Gaj T, Guo J, Kato Y, Sirk SJ, Barbas CF 3rd (2012) Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 9:805–807
Liu J, Gaj T, Yang Y, Wang N, Shui S, Kim S, Kanchiswamy CN, Kim JS, Barbas CF 3rd (2015) Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells. Nat Protoc 10:1842–1859
Gaj T, Liu J, Anderson KE, Sirk SJ, Barbas CF 3rd (2014) Protein delivery using Cys2-His2 zinc-finger domains. ACS Chem Biol 9:1662–1667
Gaj T, Liu J (2015) Direct protein delivery to mammalian cells using cell-permeable Cys2-His2 zinc-finger domains. J Vis Exp 97:e52814
Liu J, Gaj T, Wallen MC, Barbas CF 3rd (2015) Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering. Mol Ther Nucleic Acids 4:e232
Chen Z, Jaafar L, Agyekum DG, Xiao H, Wade MF, Kumaran RI, Spector DL, Bao G, Porteus MH, Dynan WS, Meiler SE (2013) Receptor-mediated delivery of engineered nucleases for genome modification. Nucleic Acids Res 41:e182
Bobis-Wozowicz S, Galla M, Alzubi J, Kuehle J, Baum C, Schambach A, Cathomen T (2014) Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery. Sci Rep 4:4656
Cai Y, Bak RO, Mikkelsen JG (2014) Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases. elife 3:e01911
Liu J, Gaj T, Patterson JT, Sirk SJ, Barbas CF 3rd (2014) Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. PLoS One 9:e85755
Ru R, Yao Y, Yu S, Yin B, Xu W, Zhao S, Qin L, Chen X (2013) Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs. Cell Regen 2:5
Kim S, Kim D, Cho SW, Kim J, Kim JS (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019
Lin S, Staahl BT, Alla RK, Doudna JA (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. elife 3:e04766
Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73–80
Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020–1027
Wang M, Zuris JA, Meng F, Rees H, Sun S, Deng P, Han Y, Gao X, Pouli D, Wu Q, Georgakoudi I, Liu DR, Xu Q (2016) Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc Natl Acad Sci U S A 113:2868–2873
Mout R, Ray M, Yesilbag Tonga G, Lee YW, Tay T, Sasaki K, Rotello VM (2017) Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano 11:2452–2458
Ha JS, Lee JS, Jeong J, Kim H, Byun J, Kim SA, Lee HJ, Chung HS, Lee JB, Ahn DR (2017) Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption. J Control Release 250:27–35
Lee K, Conboy M, Park HM, Jiang F, Kim HJ, Dewitt MA, Mackley VA, Chang K, Rao A, Skinner C, Shobha T, Mehdipour M, Liu H, Huang W-C, Lan F, Bray NL, Li S, Corn JE, Kataoka K, Doudna JA, Conboy I, Murthy N (2017) Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng 1:889–901
D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V, Lebbink RJ, Rehmann H, Geijsen N (2015) Efficient intracellular delivery of native proteins. Cell 161:674–690
Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, Pavel-Dinu M, Saxena N, Wilkens AB, Mantri S, Uchida N, Hendel A, Narla A, Majeti R, Weinberg KI, Porteus MH (2016) CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 539:384–389
Gaj T, Staahl BT, Rodrigues GM, Limsirichai P, Ekman FK, Doudna JA, Schaffer DV (2017) Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery. Nucleic Acids Res 45:e98
Rodrigues GMC, Gaj T, Adil MM, Wahba J, Rao AT, Lorbeer FK, Kulkarni RU, Diogo MM, Cabral JMS, Miller EW, Hockemeyer D, Schaffer DV (2017) Defined and scalable differentiation of human oligodendrocyte precursors from pluripotent stem cells in a 3D culture system. Stem Cell Rep 8:1770–1783
Gaj T, Schaffer DV (2016) Adeno-associated virus-mediated delivery of CRISPR-Cas systems for genome engineering in mammalian cells. Cold Spring Harb Protoc 2016:pdb prot086868
Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649:247–256
Acknowledgments
This work was supported by Natural Science Foundation of China (No. 31600686 to J.L.) and ShanghaiTech University (Startup fund to the Laboratory of ADC Chemistry).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Liu, J., Liang, Yj., Ren, Pl., Gaj, T. (2018). Manufacturing and Delivering Genome-Editing Proteins. In: Liu, J. (eds) Zinc Finger Proteins. Methods in Molecular Biology, vol 1867. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8799-3_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8799-3_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8798-6
Online ISBN: 978-1-4939-8799-3
eBook Packages: Springer Protocols