Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

Abstract The CRISPR–Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR–Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information.

[1]  Daniel G. Anderson,et al.  Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo , 2016, Nature Biotechnology.

[2]  ジョン，ジェー．キース,et al.  Engineered CRISPR-Cas9 nucleases with altered PAM specificity , 2016 .

[3]  Chris P Ponting,et al.  Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System , 2014, Cell reports.

[4]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[5]  Robert L. Judson,et al.  Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells , 2011, Nature Biotechnology.

[6]  A. Krogh,et al.  Programmed Cell Death 4 (PDCD4) Is an Important Functional Target of the MicroRNA miR-21 in Breast Cancer Cells* , 2008, Journal of Biological Chemistry.

[7]  Jian Kang,et al.  Artemin is oncogenic for human mammary carcinoma cells , 2009, Oncogene.

[8]  Martin J. Aryee,et al.  Engineered CRISPR-Cas9 nucleases with altered PAM specificities , 2015, Nature.

[9]  Chunxi Wang,et al.  Clinical and tumor significance of tropomyosin-1 expression levels in renal cell carcinoma. , 2015, Oncology reports.

[10]  J. Coselli,et al.  Up-regulation of PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome Ten) Mediates p38 MAPK Stress Signal-induced Inhibition of Insulin Signaling , 2006, Journal of Biological Chemistry.

[11]  Xiaoqiang Guo,et al.  Directing cellular information flow via CRISPR signal conductors , 2016, Nature Methods.

[12]  Dongsheng Duan,et al.  In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy , 2016, Science.

[13]  Ron Weiss,et al.  Mammalian synthetic circuits with RNA binding proteins delivered by RNA , 2015, Nature Biotechnology.

[14]  Alexander Meissner,et al.  Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. , 2010, Cell stem cell.

[15]  Z. Cai,et al.  Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells , 2014, Nature Communications.

[16]  Feng Zhang,et al.  A split-Cas9 architecture for inducible genome editing and transcription modulation , 2015, Nature Biotechnology.

[17]  Tetsushi Sakuma,et al.  Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9 , 2014, Stem cell reports.

[18]  R. Sachidanandam,et al.  High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries , 2012, Nature Methods.

[19]  David A. Scott,et al.  Rationally engineered Cas9 nucleases with improved specificity , 2015, Science.

[20]  Alexander Deiters,et al.  Optical Control of CRISPR/Cas9 Gene Editing. , 2015, Journal of the American Chemical Society.

[21]  C. Tsao,et al.  Emerging role of microRNA-21 in cancer (Review) , 2016 .

[22]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[23]  M. Batzer,et al.  The impact of retrotransposons on human genome evolution , 2009, Nature Reviews Genetics.

[24]  K. Woltjen,et al.  Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives. , 2016, Methods.

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

[26]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[27]  Shinya Yamanaka,et al.  MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells , 2016, Scientific Reports.

[28]  Y. Mukouyama,et al.  TGFβ signaling is required for sprouting lymphangiogenesis during lymphatic network development in the skin , 2013, Development.

[29]  C. Tsao,et al.  Emerging role of microRNA-21 in cancer. , 2016, Biomedical reports.

[30]  Dacheng Ma,et al.  Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells , 2016, Nature Communications.

[31]  J. Van Damme,et al.  Hypoxia enhances CXCR4 expression in human microvascular endothelial cells and human melanoma cells. , 2007, European cytokine network.

[32]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[33]  Mark A. Kay,et al.  Progress and problems with the use of viral vectors for gene therapy , 2003, Nature Reviews Genetics.

[34]  Shinya Yamanaka,et al.  Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches. , 2015, Cell stem cell.

[35]  Namritha Ravinder,et al.  Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. , 2015, Journal of biotechnology.