Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells.
Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China.
Jia
J
Liu
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.|Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA.|Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
Thomas
T
Gaj
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China.
Yifeng
Y
Yang
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China.
Nan
N
Wang
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China.
Sailan
S
Shui
Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea.
Sojung
S
Kim
Department of Genomics and Biology of Fruit Crop, Research and Innovation Centre, Fondazione Edmund Mach (FEM), San Michele all'Adige, Italy.
Chidananda Nagamangala
CN
Kanchiswamy
Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea.|Department of Chemistry, Seoul National University, Seoul, South Korea.
Jin-Soo
JS
Kim
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.|Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA.|Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
Carlos F
CF
Barbas
D002478:Cells, Cultured; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D021381:Protein Transport; D045522:Recombinases / Q000378:metabolism*; D013234:Stem Cells
D045522:Recombinases