Abstract
Genome editing using the CRISPR/Cas9 system has rapidly established itself as an essential tool in the genetic manipulation of many organisms, including human cell lines. Its application to human induced pluripotent stem cells (hiPSCs) allows for the generation of isogenic cell pairs that differ in a single genetic lesion, and therefore the identification and characterization of causal genetic variants. We describe a simple, effective approach to perform delicate manipulations of the genome of hiPSCs through delivery of Cas9 RNPs along with ssDNA oligonucleotide repair templates that can generate mutations in up to 98% of single cell clones and introduce single nucleotide changes at an efficiency of up to 40%. We describe our use of a T7 endonuclease assay to identify active guide RNAs, and a high-throughput sequencing genotyping strategy that allows the identification of correctly edited clones. We also present our experiences of generating single nucleotide changes at 15 sites, which show considerable variability between both guides and target sites in the efficiency at which such changes can be introduced.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bassett AR (2017) Editing the genome of hiPSC with CRISPR/Cas9: disease models. Mamm Genome 28:348–364
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
Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764
Shrivastav M, De Haro LP, Nickoloff JA (2008) Regulation of DNA double-strand break repair pathway choice. Cell Res 18:134–147
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
Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53
Kan Y, Ruis B, Takasugi T, Hendrickson EA (2017) Mechanisms of precise genome editing using oligonucleotide donors. Genome Res 27:1099–1111
Davis L, Maizels N (2016) Two distinct pathways support gene correction by single-stranded donors at DNA nicks. Cell Rep 17:1872–1881
Richardson CD, Kazane KR, Feng SJ, Zelin E, Bray NL, Schäfer AJ, Floor SN, Corn JE (2018) CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat Genet 50(8):1132–1139
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 (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832
Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JR, Aryee MJ, Joung JK (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481–485
Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556:57–63
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826
Hodgkins A, Farne A, Perera S, Grego T, Parry-Smith DJ, Skarnes WC, Iyer V (2015) WGE: a CRISPR database for genome engineering. Bioinformatics 31:3078–3080
Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–88
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191
Kilpinen H, Goncalves A, Leha A, Afzal V, Alasoo K, Ashford S, Bala S, Bensaddek D, Casale FP, Culley OJ, Danecek P, Faulconbridge A, Harrison PW, Kathuria A, McCarthy D, McCarthy SA, Meleckyte R, Memari Y, Moens N, Soares F, Mann A, Streeter I, Agu CA, Alderton A, Nelson R, Harper S, Patel M, White A, Patel SR, Clarke L, Halai R, Kirton CM, Kolb-Kokocinski A, Beales P, Birney E, Danovi D, Lamond AI, Ouwehand WH, Vallier L, Watt FM, Durbin R, Stegle O, Gaffney DJ (2017) Common genetic variation drives molecular heterogeneity in human iPSCs. Nature 546:370–375
Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42:e168
Bronner IF, Quail MA, Turner DJ, Swerdlow H (2014) Improved protocols for illumina sequencing. Curr Protoc Hum Genet 80:18.2.1–18.242
Kozarewa I, Turner DJ (2011) 96-plex molecular barcoding for the illumina genome analyzer. Methods Mol Biol 733:279–298
Pinello L, Canver MC, Hoban MD, Orkin SH, Kohn DB, Bauer DE, Yuan GC (2016) Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol 34:695–697
Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–548
Carlson-Stevermer J, Abdeen AA, Kohlenberg L, Goedland M, Molugu K, Lou M, Saha K (2017) Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat Commun 8:1711
Agudelo D, Duringer A, Bozoyan L, Huard CC, Carter S, Loehr J, Synodinou D, Drouin M, Salsman J, Dellaire G, Laganiere J, Doyon Y (2017) Marker-free coselection for CRISPR-driven genome editing in human cells. Nat Methods 14:615–620
Zhou Y, Liu Y, Hussmann D, Brogger P, Al-Saaidi RA, Tan S, Lin L, Petersen TS, Zhou GQ, Bross P, Aagaard L, Klein T, Ronn SG, Pedersen HD, Bolund L, Nielsen AL, Sorensen CB, Luo Y (2016) Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system. Cell Mol Life Sci 73:2543–2563
Hirano H, Gootenberg JS, Horii T, Abudayyeh OO, Kimura M, Hsu PD, Nakane T, Ishitani R, Hatada I, Zhang F, Nishimasu H, Nureki O (2016) Structure and engineering of francisella novicida Cas9. Cell 164:950–961
Ellerstrom C, Strehl R, Noaksson K, Hyllner J, Semb H (2007) Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation. Stem Cells 25:1690–1696
Thomson A, Wojtacha D, Hewitt Z, Priddle H, Sottile V, Di Domenico A, Fletcher J, Waterfall M, Corrales NL, Ansell R, McWhir J (2008) Human embryonic stem cells passaged using enzymatic methods retain a normal karyotype and express CD30. Cloning Stem Cells 10:89–106
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bruntraeger, M., Byrne, M., Long, K., Bassett, A.R. (2019). Editing the Genome of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes. In: Luo, Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9170-9_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9170-9_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9169-3
Online ISBN: 978-1-4939-9170-9
eBook Packages: Springer Protocols