リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

大学・研究所にある論文を検索できる 「Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

コピーが完了しました

URLをコピーしました

論文の公開元へ論文の公開元へ
書き出し

Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

Xu, Huaigeng Kita, Yuto Bang, Uikyu Gee, Peter Hotta, Akitsu 京都大学 DOI:10.1016/j.xpro.2021.100965

2021.12.17

概要

Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).

この論文で使われている画像

参考文献

Bamshad, M.J., Ng, S.B., Bigham, A.W., Tabor,

H.K., Emond, M.J., Nickerson, D.A., and Shendure,

J. (2011). Exome sequencing as a tool for

Mendelian disease gene discovery. Nat. Rev.

Genet. 12, 745–755. https://doi.org/10.1038/

nrg3031.

Branda, C.S., and Dymecki, S.M. (2004). Talking

about a revolution: the impact of site-specific

recombinases on genetic analyses in mice. Dev.

Cell 6, 7–28. https://doi.org/10.1016/s15345807(03)00399-x.

iPS and myogenic cells. Nucleic Acids Res. 46,

8275–8298. https://doi.org/10.1093/nar/gky548.

Doench, J.G., Hartenian, E., Graham, D.B.,

Tothova, Z., Hegde, M., Smith, I., Sullender, M.,

Ebert, B.L., Xavier, R.J., and Root, D.E. (2014).

Rational design of highly active sgRNAs for

CRISPR-Cas9-mediated gene inactivation. Nat.

Biotechnol. 32, 1262–1267. https://doi.org/10.

1038/nbt.3026.

Brinkman, E.K., Chen, T., Amendola, M., and van

Steensel, B. (2014). Easy quantitative assessment of

genome editing by sequence trace

decomposition. Nucleic Acids Res. 42, e168.

https://doi.org/10.1093/nar/gku936.

Gee, P., Lung, M.S.Y., Okuzaki, Y., Sasakawa, N.,

Iguchi, T., Makita, Y., Hozumi, H., Miura, Y., Yang,

L.F., Iwasaki, M., et al. (2020). Extracellular

nanovesicles for packaging of CRISPR-Cas9 protein

and sgRNA to induce therapeutic exon skipping.

Nat. Commun. 11, 1334. https://doi.org/10.1038/

s41467-020-14957-y.

Collins, F.S., Guyer, M.S., and Chakravarti, A.

(1997). Variations on a theme: cataloging human

DNA sequence variation. Science 278, 1580–1581.

https://doi.org/10.1126/science.278.5343.1580.

Gutschner, T., Haemmerle, M., Genovese, G.,

Draetta, G.F., and Chin, L. (2016). Post-translational

regulation of Cas9 during G1 enhances homologydirected repair. Cell Rep. 14, 1555–1566. https://

doi.org/10.1016/j.celrep.2016.01.019.

Concordet, J.P., and Haeussler, M. (2018).

CRISPOR: intuitive guide selection for CRISPR/

Cas9 genome editing experiments and screens.

Nucleic Acids Res. 46, W242–W245. https://doi.

org/10.1093/nar/gky354.

Dastidar, S., Ardui, S., Singh, K., Majumdar, D.,

Nair, N., Fu, Y., Reyon, D., Samara, E., Gerli, M.F.M.,

Klein, A.F., et al. (2018). Efficient CRISPR/Cas9mediated editing of trinucleotide repeat

expansion in myotonic dystrophy patient-derived

22

Howden, S.E., McColl, B., Glaser, A., Vadolas, J.,

Petrou, S., Little, M.H., Elefanty, A.G., and Stanley,

E.G. (2016). A Cas9 variant for efficient generation

of indel-free knockin or gene-corrected human

pluripotent stem cells. Stem Cell Reports 7,

508–517. https://doi.org/10.1016/j.stemcr.2016.07.

001.

Ishida, K., Xu, H., Sasakawa, N., Lung, M.S.Y.,

Kudryashev, J.A., Gee, P., and Hotta, A. (2018). Sitespecific randomization of the endogenous genome

STAR Protocols 2, 100965, December 17, 2021

by a regulatable CRISPR-Cas9 piggyBac system in

human cells. Sci. Rep. 8, 310. https://doi.org/10.

1038/s41598-017-18568-4.

Kagita, A., Lung, M.S.Y., Xu, H., Kita, Y., Sasakawa,

N., Iguchi, T., Ono, M., Wang, X.H., Gee, P., and

Hotta, A. (2021). Efficient ssODN-mediated

targeting by avoiding cellular inhibitory RNAs

through precomplexed CRISPR-Cas9/sgRNA

ribonucleoprotein. Stem Cell Reports 16, 985–996.

https://doi.org/10.1016/j.stemcr.2021.02.013.

Kim, S., Kim, D., Cho, S.W., Kim, J., and Kim, J.S.

(2014). Highly efficient RNA-guided genome

editing in human cells via delivery of purified Cas9

ribonucleoproteins. Genome Res. 24, 1012–1019.

https://doi.org/10.1101/gr.171322.113.

Li, H.L., Fujimoto, N., Sasakawa, N., Shirai, S.,

Ohkame, T., Sakuma, T., Tanaka, M., Amano, N.,

Watanabe, A., Sakurai, H., et al. (2015). Precise

correction of the dystrophin gene in duchenne

muscular dystrophy patient induced pluripotent

stem cells by TALEN and CRISPR-Cas9. Stem Cell

Reports 4, 143–154. https://doi.org/10.1016/j.

stemcr.2014.10.013.

Mao, Z., Bozzella, M., Seluanov, A., and

Gorbunova, V. (2008). DNA repair by

nonhomologous end joining and homologous

recombination during cell cycle in human cells. Cell

Cycle 7, 2902–2906. https://doi.org/10.4161/cc.7.

18.6679.

Miyaoka, Y., Berman, J.R., Cooper, S.B., Mayerl,

S.J., Chan, A.H., Zhang, B., Karlin-Neumann, G.A.,

A Self-archived copy in

Kyoto University Research Information Repository

https://repository.kulib.kyoto-u.ac.jp

ll

Protocol

and Conklin, B.R. (2016). Systematic quantification

of HDR and NHEJ reveals effects of locus, nuclease,

and cell type on genome-editing. Sci. Rep. 6,

23549. https://doi.org/10.1038/srep23549.

Naito, Y., Hino, K., Bono, H., and Ui-Tei, K. (2015).

CRISPRdirect: software for designing CRISPR/Cas

guide RNA with reduced off-target sites.

Bioinformatics 31, 1120–1123. https://doi.org/10.

1093/bioinformatics/btu743.

Nakagawa, M., Taniguchi, Y., Senda, S., Takizawa,

N., Ichisaka, T., Asano, K., Morizane, A., Doi, D.,

Takahashi, J., Nishizawa, M., et al. (2014). A novel

efficient feeder-free culture system for the

derivation of human induced pluripotent stem

cells. Sci. Rep. 4, 3594. https://doi.org/10.1038/

srep03594.

Okita, K., Matsumura, Y., Sato, Y., Okada, A.,

Morizane, A., Okamoto, S., Hong, H., Nakagawa,

M., Tanabe, K., Tezuka, K., et al. (2011). A more

efficient method to generate integration-free

human iPS cells. Nat. Methods 8, 409–412. https://

doi.org/10.1038/nmeth.1591.

Paix, A., Folkmann, A., Goldman, D.H., Kulaga, H.,

Grzelak, M.J., Rasoloson, D., Paidemarry, S., Green,

R., Reed, R.R., and Seydoux, G. (2017). Precision

genome editing using synthesis-dependent repair

of Cas9-induced DNA breaks. Proc. Natl. Acad. Sci.

U S A 114, E10745–E10754. https://doi.org/10.

1073/pnas.1711979114.

Paquet, D., Kwart, D., Chen, A., Sproul, A., Jacob,

S., Teo, S., Olsen, K.M., Gregg, A., Noggle, S., and

Tessier-Lavigne, M. (2016). Efficient introduction of

specific homozygous and heterozygous mutations

using CRISPR/Cas9. Nature 533, 125–129. https://

doi.org/10.1038/nature17664.

OPEN ACCESS

Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott,

D.A., and Zhang, F. (2013). Genome engineering

using the CRISPR-Cas9 system. Nat. Protoc. 8,

2281–2308. https://doi.org/10.1038/nprot.2013.

143.

Richardson, C.D., Ray, G.J., DeWitt, M.A., Curie,

G.L., and Corn, J.E. (2016). Enhancing homologydirected genome editing by catalytically active and

inactive CRISPR-Cas9 using asymmetric donor

DNA. Nat. Biotechnol. 34, 339–344. https://doi.

org/10.1038/nbt.3481.

Sauer, B., and Henderson, N. (1988). Site-specific

DNA recombination in mammalian cells by the Cre

recombinase of bacteriophage P1. Proc. Natl.

Acad. Sci. U S A 85, 5166–5170. https://doi.org/10.

1073/pnas.85.14.5166.

Soldner, F., and Jaenisch, R. (2018). Stem cells,

genome editing, and the path to translational

medicine. Cell 175, 615–632. https://doi.org/10.

1016/j.cell.2018.09.010.

Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J.,

Faircloth, B.C., Remm, M., and Rozen, S.G. (2012).

Primer3—new capabilities and interfaces. Nucleic

Acids Res. 40, e115. https://doi.org/10.1093/nar/

gks596.

Wang, T., Wei, J.J., Sabatini, D.M., and Lander, E.S.

(2014). Genetic screens in human cells using the

CRISPR-Cas9 system. Science 343, 80–84. https://

doi.org/10.1126/science.1246981.

Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A.,

Matsumura, M., Wataya, T., Takahashi, J.B.,

Nishikawa, S., Nishikawa, S., Muguruma, K., and

Sasai, Y. (2007). A ROCK inhibitor permits survival

of dissociated human embryonic stem cells. Nat.

Biotechnol. 25, 681–686. https://doi.org/10.1038/

nbt1310.

Xu, H., Wang, B., Ono, M., Kagita, A., Fujii, K.,

Sasakawa, N., Ueda, T., Gee, P., Nishikawa, M.,

Nomura, M., et al. (2019). Targeted Disruption of

HLA Genes via CRISPR-Cas9 Generates iPSCs with

Enhanced Immune Compatibility. Cell Stem Cell

24, 566–578. https://doi.org/10.1016/j.stem.2019.

02.005.

Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I.,

Rozen, S., and Madden, T.L. (2012). Primer-BLAST:

a tool to design target-specific primers for

polymerase chain reaction. BMC Bioinformatics

13, 134. https://doi.org/10.1186/1471-2105-13134.

Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O.,

Slaymaker, I.M., Makarova, K.S., Essletzbichler, P.,

Volz, S.E., Joung, J., van der Oost, J., Regev, A.,

et al. (2015). Cpf1 is a single RNA-guided

endonuclease of a class 2 CRISPR-Cas system. Cell

163, 759–771. https://doi.org/10.1016/j.cell.2015.

09.038.

Zhong, A., Li, M., and Zhou, T. (2020). Protocol

for the generation of human pluripotent reporter

cell lines using CRISPR/Cas9. STAR Protoc. 1,

100052. https://doi.org/10.1016/j.xpro.2020.

100052.

Zuris, J.A., Thompson, D.B., Shu, Y., Guilinger, J.P.,

Bessen, J.L., Hu, J.H., Maeder, M.L., Joung, J.K.,

Chen, Z.Y., and Liu, D.R. (2015). Cationic lipidmediated delivery of proteins enables efficient

protein-based genome editing in vitro and in vivo.

Nat. Biotechnol. 33, 73–80. https://doi.org/10.

1038/nbt.3081.

STAR Protocols 2, 100965, December 17, 2021

23

...

参考文献をもっと見る

全国の大学の
卒論・修論・学位論文

一発検索!

この論文の関連論文を見る