Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation

被引：0

作者：

Kiesler, Patricia ^{[1
]}

Lee, Stella S. ^{[2
]}

Norris, Alexis L. ^{[2
]}

Miller, Mayumi F. ^{[3
]}

Mercado, Carlo J. ^{[2
]}

Moyer, Adam L. ^{[2
]}

Maragh, Samantha ^{[1
]}

机构：

[1] NIST, Biosyst & Biomat Div, Gaithersburg, MD 20899 USA

[2] US FDA, Ctr Vet Med, Off New Anim Drug Evaluat, Rockville, MD 20855 USA

[3] US FDA, Ctr Vet Med, Off Appl Sci, Laurel, MD 20708 USA

来源：

STAR PROTOCOLS | 2024年 / 5卷 / 04期

关键词：

samantha.ma ragh@nist; CRISPR-Cas9; system; RNP complex; functionality; assessing on-target; editing efficiency; PORCINE; DESIGN;

D O I：

10.1016/j.xpro.2024.103385

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, , delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.

引用

页数：19

共 25 条

[1] Design and testing of a humanized porcine donor for xenotransplantation [J].

Anand, Ranjith P. ;

Layer, Jacob V. ;

Heja, David ;

Hirose, Takayuki ;

Lassiter, Grace ;

Firl, Daniel J. ;

Paragas, Violette B. ;

Akkad, Adam ;

Chhangawala, Sagar ;

Colvin, Robert B. ;

Ernst, Russell J. ;

Esch, Nicholas ;

Getchell, Kristen ;

Griffin, Alexandra K. ;

Guo, Xiaoyun ;

Hall, Katherine C. ;

Hamilton, Paula ;

Kalekar, Lokesh A. ;

Kan, Yinan ;

Karadagi, Ahmad ;

Li, Feng ;

Low, Susan C. ;

Matheson, Rudy ;

Nehring, Claudia ;

Otsuka, Ryo ;

Pandelakis, Matthew ;

Policastro, Robert A. ;

Pols, Rebecca ;

Queiroz, Luis ;

Rosales, Ivy A. ;

Serkin, William T. ;

Stiede, Kathryn ;

Tomosugi, Toshihide ;

Xue, Yongqiang ;

Zentner, Gabriel E. ;

Angeles-Albores, David ;

Chris Chao, J. ;

Crabtree, Juliet N. ;

Harken, Sierra ;

Hinkle, Nicole ;

Lemos, Tania ;

Li, Mailin ;

Pantano, Lorena ;

Stevens, Denise ;

Subedar, Omar D. ;

Tan, Xiaoqing ;

Yin, Shiyi ;

Anwar, Imran J. ;

Aufhauser, David ;

Capuano, Saverio .

NATURE, 2023, 622 (7982) :393-401

[2] Tools for experimental and computational analyses of off-target editing by programmable nucleases [J].

Bao, X. Robert ;

Pan, Yidan ;

Lee, Ciaran M. ;

Davis, Timothy H. ;

Bao, Gang .

NATURE PROTOCOLS, 2021, 16 (01) :10-26

[3] Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR-Edited Sanger sequencing Data [J].

Bloh, Kevin ;

Kanchana, Rohan ;

Bialk, Pawel ;

Banas, Kelly ;

Zhang, Zugui ;

Yoo, Byung-Chun ;

Kmiec, Eric B. .

CRISPR JOURNAL, 2021, 4 (01) :120-131

[4] Easy quantitative assessment of genome editing by sequence trace decomposition [J].

Brinkman, Eva K. ;

Chen, Tao ;

Amendola, Mario ;

van Steensel, Bas .

NUCLEIC ACIDS RESEARCH, 2014, 42 (22)

[5] Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas [J].

Burger, Brian T. ;

Beaton, Benjamin P. ;

Campbell, Matthew A. ;

Brett, Benjamin T. ;

Rohrer, Melissa S. ;

Plummer, Sarah ;

Barnes, Dylan ;

Jiang, Ke ;

Naswa, Sudhir ;

Lange, Jeremy ;

Ott, Alina ;

Alger, Elizabeth ;

Rincon, Gonzalo ;

Rounsley, Steven ;

Betthauser, Jeff ;

Mtango, Namdori R. ;

Benne, Joshua A. ;

Hammerand, Jessica ;

Durfee, Codie J. ;

Rotolo, Marisa L. ;

Cameron, Peter ;

Lied, Alexandra M. ;

Irby, Matthew J. ;

Nyer, David B. ;

Fuller, Chris K. ;

Gradia, Scott ;

Kanner, Steven B. ;

Park, Ki-Eun ;

Waters, Jerel ;

Simpson, Sean ;

Telugu, Bhanu P. ;

Salgado, Brianna C. ;

Brandariz-Nunez, Alberto ;

Rowland, Raymond R. R. ;

Culbertson, Matt ;

Rice, Elena ;

Cigan, A. Mark .

CRISPR JOURNAL, 2024, 7 (01) :12-28

[6] Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets [J].

Chen, Qi ;

Gauger, Phillip ;

Stafne, Molly ;

Thomas, Joseph ;

Arruda, Paulo ;

Burrough, Eric ;

Madson, Darin ;

Brodie, Joseph ;

Magstadt, Drew ;

Derscheid, Rachel ;

Welch, Michael ;

Zhang, Jianqiang .

VIROLOGY, 2015, 482 :51-59

[7] Inference of CRISPR Edits from Sanger Trace Data [J].

Conant, David ;

Hsiau, Tim ;

Rossi, Nicholas ;

Oki, Jennifer ;

Maures, Travis ;

Waite, Kelsey ;

Yang, Joyce ;

Joshi, Sahil ;

Kelso, Reed ;

Holden, Kevin ;

Enzmann, Brittany L. ;

Stoner, Rich .

CRISPR JOURNAL, 2022, 5 (01) :123-130

[8] CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing [J].

Dehairs, Jonas ;

Talebi, Ali ;

Cherifi, Yacine ;

Swinnen, Johannes V. .

SCIENTIFIC REPORTS, 2016, 6

[9] N-Acetyl-L-Cysteine Ameliorates BPAF-Induced Porcine Sertoli Cell Apoptosis and Cell Cycle Arrest via Inhibiting the ROS Level [J].

Feng, Yue ;

Wu, Junjing ;

Lei, Runyu ;

Zhang, Yu ;

Qiao, Mu ;

Zhou, Jiawei ;

Xu, Zhong ;

Li, Zipeng ;

Sun, Hua ;

Peng, Xianwen ;

Mei, Shuqi .

TOXICS, 2023, 11 (11)

[10] Computational approaches for effective CRISPR guide RNA design and evaluation [J].

Liu, Guanqing ;

Zhang, Yong ;

Zhang, Tao .

COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL, 2020, 18 (18) :35-44

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