An Efficient CRISPR-Cas9 DNA Editing Methodology Applicable for iPSC Disease Modelling

The capability to generate induced pluripotent cell (iPSC) lines, in combination with the CRISPR-Cas9 DNA editing technology, offers great promise to understand the underlying genetic mechanisms of human disease. However, technical impediments including, but not limited to, low transfection efficiency, single-cell survival, and high clonal heterogeneity, limit the potential of these techniques. Here we provide an efficient methodology addressing these challenges, resulting in high transfection efficiency exceeding 97% with an increased single cell clone survival rate (up to 70%). These enhancements were accompanied by a high editing efficiency in the range of 48.6 to 57.5%, comparable to existing methods.

[1]  K. Brennand,et al.  Integration of CRISPR-engineering and hiPSC-based models of psychiatric genomics , 2020, Molecular and Cellular Neuroscience.

[2]  M. Kampmann.  CRISPR-based functional genomics for neurological disease , 2020, Nature Reviews Neurology.

[3]  Hua Zhu,et al.  A simple, quick, and efficient CRISPR/Cas9 genome editing method for human induced pluripotent stem cells , 2020, Acta Pharmacologica Sinica.

[4]  D. Avramopoulos,et al.  Modeling Psychiatric Disorder Biology with Stem Cells , 2020, Current Psychiatry Reports.

[5]  D. Spengler,et al.  Focus on Causality in ESC/iPSC-Based Modeling of Psychiatric Disorders , 2020, Cells.

[6]  Nicholas A. Rossi,et al.  Inference of CRISPR Edits from Sanger Trace Data , 2019, bioRxiv.

[7]  T. Enoki,et al.  Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs , 2019, Scientific Reports.

[8]  Amar M. Singh.  An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells , 2019, Front. Cell Dev. Biol..

[9]  M. Bellgrove,et al.  Generation of four iPSC lines from peripheral blood mononuclear cells (PBMCs) of an attention deficit hyperactivity disorder (ADHD) individual and a healthy sibling in an Australia-Caucasian family. , 2019, Stem cell research.

[10]  Jian Chen,et al.  Efficient homology-directed gene editing by CRISPR/Cas9 in human stem and primary cells using tube electroporation , 2018, Scientific Reports.

[11]  Robert Langer,et al.  Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts. , 2018, Chemical reviews.

[12]  Gang Bao,et al.  A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells , 2018, Nature Medicine.

[13]  Gregory McAllister,et al.  p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells , 2018, Nature Medicine.

[14]  A. Chen-Plotkin,et al.  The Post-GWAS Era: From Association to Function. , 2018, American journal of human genetics.

[15]  R. Kühn,et al.  Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9. , 2017, Methods.

[16]  G. Church,et al.  Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies , 2016, Nature Protocols.

[17]  Xiao-Hui Zhang,et al.  Off-target Effects in CRISPR/Cas9-mediated Genome Engineering , 2015, Molecular therapy. Nucleic acids.

[18]  Andrew J Petersen,et al.  Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9. , 2015, Cell stem cell.

[19]  J. Keith Joung,et al.  Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo , 2014, Nature Biotechnology.

[20]  Daesik Kim,et al.  Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins , 2014, Genome research.

[21]  B. Conklin,et al.  Isolation of single-base genome-edited human iPS cells without antibiotic selection , 2014, Nature Methods.

[22]  P. Cahan,et al.  Origins and implications of pluripotent stem cell variability and heterogeneity , 2013, Nature Reviews Molecular Cell Biology.

[23]  Susan Lindquist,et al.  Generation of Isogenic Pluripotent Stem Cells Differing Exclusively at Two Early Onset Parkinson Point Mutations , 2011, Cell.

[24]  Yoshiki Sasai,et al.  Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. , 2010, Cell stem cell.

[25]  J. Thomson,et al.  Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. , 2010, Cell stem cell.

[26]  J. Navarro,et al.  Electron microscopy reveals the presence of viruses in mouse embryonic fibroblasts but neither in human embryonic fibroblasts nor in human mesenchymal cells used for hESC maintenance: toward an implementation of microbiological quality assurance program in stem cell banks. , 2008, Cloning and stem cells.

[27]  Jack A. M. Leunissen,et al.  Turning CFCs into salt. , 1996, Nucleic Acids Res..

[28]  Susana Castro-Obregon,et al.  New non-viral method for gene transfer into primary cells. , 2004, Methods.