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Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.
Jao LE, Wente SR, Chen W
(2013) Proc Natl Acad Sci U S A 110: 13904-9
MeSH Terms: Animals, Breeding, Deoxyribonucleases, Gene Knockout Techniques, Genetic Engineering, Genome, Inverted Repeat Sequences, Mutagenesis, Site-Directed, Phenotype, Zebrafish
Show Abstract · Added March 7, 2014
A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
0 Communities
2 Members
0 Resources
10 MeSH Terms
Multiplex genome engineering using CRISPR/Cas systems.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F
(2013) Science 339: 819-23
MeSH Terms: Animals, Base Sequence, CRISPR-Cas Systems, DNA, DNA Cleavage, Genetic Engineering, Genetic Loci, Genome, Humans, Inverted Repeat Sequences, Mice, Microarray Analysis, Molecular Sequence Data, Mutagenesis, RNA, Recombinational DNA Repair, Streptococcus pyogenes
Show Abstract · Added August 13, 2013
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
0 Communities
0 Members
1 Resources
17 MeSH Terms
Increased cell migration and plasticity in Nrf2-deficient cancer cell lines.
Rachakonda G, Sekhar KR, Jowhar D, Samson PC, Wikswo JP, Beauchamp RD, Datta PK, Freeman ML
(2010) Oncogene 29: 3703-14
MeSH Terms: Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Survival, Humans, Inverted Repeat Sequences, NF-E2-Related Factor 2, Neoplasms, Phosphorylation, RNA Interference, RNA, Small Interfering, Reactive Oxygen Species, Smad Proteins, Transcription, Genetic, ras Proteins
Show Abstract · Added May 3, 2013
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression is deregulated in many cancers. Genetic and biochemical approaches coupled with functional assays in cultured cells were used to explore the consequences of Nrf2 repression. Nrf2 suppression by Keap1-directed ubiquitylation or the expression of independent short hairpin RNA (shRNA)/siRNA sequences enhanced cellular levels of reactive oxygen species, Smad-dependent tumor cell motility and growth in soft agar. Loss of Nrf2 was accompanied by concomitant Smad linker region/C-terminus phosphorylation, induction of the E-cadherin transcriptional repressor Slug and suppression of the cell-cell adhesion protein E-cadherin. Ectopic expression of the wildtype but not dominant-negative Nrf2 suppressed the activity of a synthetic transforming growth factor-beta1-responsive CAGA-directed luciferase reporter. shRNA knock-down of Nrf2 enhanced the activity of the synthetic CAGA reporter, as well as the expression of the endogenous Smad target gene plasminogen activator inhibitor-1. Finally, we found that Nrf2/Smad3/Smad4 formed an immunoprecipitable nuclear complex. Thus, loss of Nrf2 increased R-Smad phosphorylation and R-Smad signaling, supporting the hypothesis that loss of Nrf2 in an oncogenic context-dependent manner can enhance cellular plasticity and motility, in part by using transforming growth factor-beta/Smad signaling.
1 Communities
4 Members
0 Resources
15 MeSH Terms
Specific cross-talk between epidermal growth factor receptor and integrin alphavbeta5 promotes carcinoma cell invasion and metastasis.
Ricono JM, Huang M, Barnes LA, Lau SK, Weis SM, Schlaepfer DD, Hanks SK, Cheresh DA
(2009) Cancer Res 69: 1383-91
MeSH Terms: Animals, Carcinoma, Cell Movement, Chick Embryo, DNA Primers, Epidermal Growth Factor, ErbB Receptors, Gene Knockdown Techniques, Humans, Inverted Repeat Sequences, Lung, Lung Neoplasms, Mutation, Neoplasm Invasiveness, Neoplasm Metastasis, Pancreatic Neoplasms, Polymerase Chain Reaction, RNA, Neoplasm, Receptor Cross-Talk, Receptors, Vitronectin, Tumor Cells, Cultured
Show Abstract · Added January 20, 2015
Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin alpha(v)beta(5). Here, we show that EGF stimulates metastasis of carcinoma cells via a Src-dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and alpha(v)beta(5) activation. This pathway induces alpha(v)beta(5)-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease.
0 Communities
1 Members
0 Resources
21 MeSH Terms