Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells

2015 ◽  
Vol 10 (11) ◽  
pp. 1842-1859 ◽  
Author(s):  
Jia Liu ◽  
Thomas Gaj ◽  
Yifeng Yang ◽  
Nan Wang ◽  
Sailan Shui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Primary Cells ◽  
Human Stem Cells ◽  
Efficient Delivery

scholarly journals Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

2017 ◽  
Author(s):  
Brock Roberts ◽  
Amanda Haupt ◽  
Andrew Tucker ◽  
Tanya Grancharova ◽  
Joy Arakaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quality Control ◽  
Genome Editing ◽  
Cell Biology ◽  
Cellular Structures ◽  
Human Stem Cells ◽  
Alpha Tubulin ◽  
Junction Protein ◽  
Endogenous Proteins ◽  
Clonal Lines

AbstractWe present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ∼25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

scholarly journals Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

2017 ◽  
Author(s):  
Philippe E. Mangeot ◽  
Valérie Risson ◽  
Floriane Fusil ◽  
Aline Marnef ◽  
Emilie Laurent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Target Cells ◽  
Mouse Bone Marrow ◽  
Primary Cells ◽  
Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

scholarly journals “Ancestralization” of human pluripotent stem cells by multiplexed precise genome editing

2018 ◽  
Author(s):  
Stephan Riesenberg ◽  
Tomislav Maricic ◽  
Svante Pääbo
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Nucleotide Substitution ◽  
Human Stem Cells ◽  
Animal Cells ◽  
Isolated Cells ◽  
Dependent Protein Kinase ◽  
Drastic Increase ◽  
Dependent Protein

We show that inactivation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) results in a drastic increase in efficiency of precise genome editing with CRISPR enzymes in human stem cells, allowing up to 79% of chromosomes to carry an intended nucleotide substitution when a single genomic site is targeted. When three different genes are simultaneously targeted, 12% of the isolated cells carry the targeted amino acid-changing substitutions in homozygous forms. These substitutions represent the first step towards resurrecting the proteome ancestral to Neandertals and modern humans. DNA-PKcs inactivation will greatly facilitate multiplexed precise genome editing in animal cells.

Genome editing: a robust technology for human stem cells

2017 ◽  
Vol 74 (18) ◽  
pp. 3335-3346 ◽  
Author(s):  
Arun Pandian Chandrasekaran ◽  
Minjung Song ◽  
Suresh Ramakrishna
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Human Stem Cells

Biallelic genome editing of human stem cells at scale

2016 ◽  
Author(s):  
Manousos Koutsourakis ◽  
Wendy Bushel ◽  
William C. Skarnes
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Human Stem Cells

scholarly journals CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects

2017 ◽  
Vol 9 ◽  
pp. 230-241 ◽  
Author(s):  
Zhao Zhang ◽  
Yuelin Zhang ◽  
Fei Gao ◽  
Shuo Han ◽  
Kathryn S. Cheah ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Current Status ◽  
Human Stem Cells ◽  
Future Prospects
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