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Cell - One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

Cell - One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering | Multi- gene | Scoop.it
#CRISPR/Cas: multiplex generation of mice with several mutated genes http://t.co/t8LI50O0vM via @GenomeEngineer @NatureBiotech
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Genome Biology | Abstract | CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a CRISPR locus between repeats, forming arrays of repeat-spacer units.
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Cancer translocations in human cells induced by zinc finger and TALE nucleases - Genome Research

Piganeau et al, 2013

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes which act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this report, we set out to use genome editing with zinc finger (ZFN) and tale effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and Anaplastic Large Cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions from the formation of Ewing sarcoma and ALCL translocations. Breakpoint junctions recovered after ZFN cleavage in human ES-cell derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.

 

 


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Transgenics: A new breed - Nature.com

Transgenics: A new breed - Nature.com | Multi- gene | Scoop.it
Nature.com
Transgenics: A new breed
Nature.com
For example, enzymes called transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) can cut DNA at specific points chosen by the experimenter.
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Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.


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High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos

High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos | Multi- gene | Scoop.it

Suzuki et al., 2013

 

Recently, gene editing with transcription activator-like effector nucleases (TALENs) has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr) and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.


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TALENs and ZFNs are associated with different mutation signatures - Nature Methods

http://www.nature.com/nmeth/journal/v10/n3/full/nmeth.2364.html?WT.ec_id=NMETH-201303

 

Kim 2013, Nature Methods

 

Zinc-finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs) are of great interest for genome engineering in higher eukaryotic cells and organisms1, 2, 3, 4, 5. These enzymes contain the same FokI nuclease domain and induce site-specific DNA cleavage; the repair of this broken DNA via error-prone nonhomologous end joining gives rise to small insertions and deletions at the cleavage site, often disrupting genetic information. We have observed that, despite their similarities, ZFNs and TALENs are associated with different mutation patterns. We first compared ZFN and TALEN mutation signatures reported in the literature. We calculated the frequencies of insertions, deletions and complex patterns that include both insertions and deletions at target sites in mammalian cells, mammalian and nonmammalian organisms, and plants. Our analysis included a total of 1,456 mutant sequences at 122 target sites reported in 43 independent studies.


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TALEN-based Gene Correction for Epidermolysis Bullosa - Molecular Therapy

(via T. Lahaye, thanks)

Osborn et al, 2013

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10 kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.


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Boris Skryabin's curator insight, April 3, 2013 3:54 AM

New technology for medicine is emerging

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The New Genetic Engineering Toolbox - BioTechniques

(via T. Lahaye, thanks!)

comparison of TAL effectors, CRISPR/Cas9 and zinc fingers


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Addgene: Qi Lab CRISPR Page

Addgene: Qi Lab CRISPR Page | Multi- gene | Scoop.it

Qi Lab CRISPR Plasmids Available from Addgene

 

A system to regulate a specific gene's expression based on the Type II bacterial CRISPR system for use in either human cells or bacterial cells that is called CRISPR Interference (CRISPRi). For use in human cells a human codon-optimized but enzymatically dead Cas9 nuclease can be targeted to a DNA sequence using a chimeric gRNA. The inactive Cas9/gRNA complex bound to DNA has been shown to interfere with transcriptional elongation, RNA polymerase binding and transcription factor binding. A BFP-Cas9 (enzymatically inactive) fusion construct was also created to help determine successful transformants. For use in bacteria a catalytically dead Cas9 expression plasmid and separate gRNA expression plasmid were also created. In addition, a wild type bacterial Cas9 allows for genome editing in bacterial cells.

 

These plasmids are described in:

Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression.Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.. PUBMED.

 


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Rescooped by Christian Faltado Cantos from Plant Breeding and Genomics News
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BMC Biotechnology | Abstract | Less is more: strategies to remove marker genes from transgenic plants

Selectable marker genes (SMGs) and selection agents are useful tools in the production of transgenic plants by selecting transformed cells from a matrix consisting of of mostly untransformed cells. Most SMGs express protein products that confer antibiotic- or herbicide resistance traits, and typically reside in the end product of genetically-modified (GM) plants. The presence of these genes in GM plants, and subsequently in food, feed and the environment, are of concern and subject to special government regulation in many countries. The presence of SMGs in GM plants might also, in some cases, result in a metabolic burden for the host plants. Their use also prevents the re-use of the same SMG when a second transformation scheme is needed to be performed on the transgenic host. In recent years, several strategies have been developed to remove SMGs from GM products while retaining the transgenes of interest. This review describes the existing strategies for SMG removal, including the implementation of site specific recombination systems, TALENs and ZFNs. This review discusses the advantages and disadvantages of existing SMG-removal strategies and explores possible future research directions for SMG removal including emerging technologies for increased precision for genome modification.


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CiteULike: CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

Quan Zhang, Mina Rho, Haixu Tang, Thomas Doak, Yuzhen Ye. Genome Biology, Vol. 14, No. 4. (2013), R40, doi:10.1186/gb-2013-14-4-r40.
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PLOS ONE: Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs)

PLOS ONE: Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs) | Multi- gene | Scoop.it

(suggested by Tom Schreiber, thx)

Hu et al, 2013

MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise control over the expression of critical protein coding genes.

Despite significant progress in our understanding of how miRNAs function in mice, there remains a fundamental need to be able to target and edit miRNA genes in the human genome. Here, we report a novel approach to disrupting human miRNA genes ex vivo using engineered TAL-effector (TALE) proteins to function as nucleases (TALENs) that specifically target and disrupt human miRNA genes. We demonstrate that functional TALEN pairs can be designed to enable disruption of miRNA seed regions, or removal of entire hairpin sequences, and use this approach to successfully target several physiologically relevant human miRNAs including miR-155*, miR-155, miR-146a and miR-125b.

This technology will allow for a substantially improved capacity to study the regulation and function of miRNAs in human cells, and could be developed into a strategic means by which miRNAs can be targeted therapeutically during human disease.


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Nature:细菌利用CRISPR/Cas系统躲避宿主免疫系统 - 生物谷 (新闻发布)

Nature:细菌利用CRISPR/Cas系统躲避宿主免疫系统 - 生物谷 (新闻发布) | Multi- gene | Scoop.it
Nature:细菌利用CRISPR/Cas系统躲避宿主免疫系统
生物谷 (新闻发布)
成簇的规律间隔性短回文重复序列(clustered regularly interspaced short palindromic repeat sequences, CRISPR)是细菌用来抵抗病毒的一种基因系统.在一项新的研究中,来自美国埃默里大学的研究人员发现CRISPR参 ...
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One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering | Multi- gene | Scoop.it

Wang et al., 2013, Cell

 

Highlights

---------------

> CRISPR/Cas9-mediated simultaneous targeting of five genes in mES cells

> Generation of Tet1/Tet2 double-mutant mice in one step

> Generation of Tet1/Tet2 double-mutant mice with predefined mutations in one step

 

Summary

--------------

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.


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wb's curator insight, May 6, 2013 10:31 AM

amazing.....

Rescooped by Christian Faltado Cantos from CRISPR-Cas System for Eukaryotic Genome Engineering
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Addgene: Qi Lab CRISPR Page

Addgene: Qi Lab CRISPR Page | Multi- gene | Scoop.it

Qi Lab CRISPR Plasmids Available from Addgene

 

A system to regulate a specific gene's expression based on the Type II bacterial CRISPR system for use in either human cells or bacterial cells that is called CRISPR Interference (CRISPRi). For use in human cells a human codon-optimized but enzymatically dead Cas9 nuclease can be targeted to a DNA sequence using a chimeric gRNA. The inactive Cas9/gRNA complex bound to DNA has been shown to interfere with transcriptional elongation, RNA polymerase binding and transcription factor binding. A BFP-Cas9 (enzymatically inactive) fusion construct was also created to help determine successful transformants. For use in bacteria a catalytically dead Cas9 expression plasmid and separate gRNA expression plasmid were also created. In addition, a wild type bacterial Cas9 allows for genome editing in bacterial cells.

 

These plasmids are described in:

Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression.Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.. PUBMED.

 


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Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems | Multi- gene | Scoop.it

Via Gerd Moe-Behrens
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Gerd Moe-Behrens's curator insight, March 25, 2013 7:51 PM

 by
James E. DiCarlo, Julie E. Norville, Prashant Mali, Xavier Rios, John Aach2 and George M. Church

"Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast."

http://bit.ly/109hfvH

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A CRISPR/Cas system mediates bacterial innate immune evasion and virulence

A CRISPR/Cas system mediates bacterial innate immune evasion and virulence | Multi- gene | Scoop.it

Sampson et al., Nature

 

CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids1, 2, 3, 4. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation5, 6, 7, 8. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes9, 10. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens11, 12, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.


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TALENs: Customizable Molecular DNA Scissors for Genome Engineering of Plants - Journal of Genetics and Genomics

(via T. Lahaye, thanks!)

Chen & Gao 2013

 

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs) consisting of an engineered specific transcription activator-like effector (TALE) DNA binding domain and a FokI cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement.


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TALEN or Cas9 -- rapid, efficient and specific choices for genomic modifications -Journal of Genetics and Genomics

Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics, including biotechnology, drug development, gene therapy and synthetic biology. However, the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency, time consuming and laborious manipulation, and off-targeting problems. Recent discoveries of TALEs (transcription activator-like effectors) coding system and CRISPR (clusters of regularly interspaced short palindromic repeats) immune system in bacteria have enabled the development of customized TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types, including human cells, and different model organisms at a very high efficiency and specificity. In this review, we first briefly summarize the development and applications of TALENs and CRISPR/Cas9 mediated genome editing technologies; compare the advantages and constraints of each method; particularly, discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy; finally, propose the future directions and perspectives for readers to make the choices.


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CRISPR helps some bacteria evade mammalian immune system - News-Medical.net

CRISPR helps some bacteria evade mammalian immune system
News-Medical.net
CRISPR, a system of genes that bacteria use to defend themselves against viruses, has been found to be involved in helping some bacteria evade the mammalian immune system.
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Compact designer TALENs for efficient genome engineering - Nature Comm.

Compact designer TALENs for efficient genome engineering - Nature Comm. | Multi- gene | Scoop.it

(via T. Lahaye, thanks!)

Beurdeley et al, 2013

Transcription activator-like effector nucleases are readily targetable ‘molecular scissors’ for genome engineering applications. These artificial nucleases offer high specificity coupled with simplicity in design that results from the ability to serially chain transcription activator-like effector repeat arrays to target individual DNA bases. However, these benefits come at the cost of an appreciably large multimeric protein complex, in which DNA cleavage is governed by the nonspecific FokI nuclease domain. Here we report a significant improvement to the standard transcription activator-like effector nuclease architecture by leveraging the partially specific I-TevI catalytic domain to create a new class of monomeric, DNA-cleaving enzymes. In vivo yeast, plant and mammalian cell assays demonstrate that the half-size, single-polypeptide compact transcription activator-like effector nucleases exhibit overall activity and specificity comparable to currently available designer nucleases. In addition, we harness the catalytic mechanism of I-TevI to generate novel compact transcription activator-like effector nuclease-based nicking enzymes that display a greater than 25-fold increase in relative targeted gene correction efficacy.


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High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases -Nucl. Acids Res.

(via T. Lahaye, thanks)

Qiu et al, 2013

Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.


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