Abstract
Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.
Highlights
Human induced pluripotent stem cells generated by reprogramming of somatic cells with four transcription factors (Oct3/4, Klf4, Sox2, and c-Myc) have properties similar to those of humanInt
We obtained induced pluripotent stem (iPS) cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) at extremely high efficiency: 63% of transfected clones were disrupted in both DNMT3B alleles
A previous study of the specificity of clustered regularly interspaced short palindromic repeats (CRISPR) suggested that the DNA target site must perfectly match the protospacer-adjacent motif (PAM) sequence (NGG) and the 12 bp seed sequence at the 3' end of the small guide RNA (sgRNA) [11]; the remaining bases are believed to be less important
Summary
Human induced pluripotent stem (iPS) cells generated by reprogramming of somatic cells with four transcription factors (Oct3/4, Klf, Sox, and c-Myc) have properties similar to those of human. Zinc-finger nucleases (ZFNs) and transcription activator-like endonucleases (TALENs) have been applied to gene manipulation of human iPS cells [3,4,5] Both ZFNs and TALENs require the design of DNA-binding proteins and complicated construction of plasmids for expression of these proteins, making these methods time-consuming and labor-intensive. Immunodeficiency, centromeric region instability and facial anomalies syndrome (ICF) is a very rare autosomal recessive disorder characterized by facial dysmorphism, immunoglobulin deficiency, and branching of chromosomes 1, 9, and 16 after phytohemagglutinin (PHA) stimulation of lymphocytes. This syndrome is caused by mutations in DNA methyltransferase 3B (DNMT3B) [13,14,15]. Our results suggest that the CRISPR system is extremely efficient and useful for genome engineering of human iPS cells
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