Name: crispr/cas9-mediated targeted integration in vivo using a homology-mediated end joining-based strategy
Uploaded: 2018-03-12T14:00:00
Description: Watch this Scientific Journal Video about CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy at JoVE.com

This work was supported by CAS Strategic Priority Research Program (XDB02050007, XDA01010409), the National Hightech R&#38;D Program (863 Program; 2015AA020307), the National Natural Science Foundation of China (NSFC grants 31522037, 31500825, 31571509, 31522038), China Youth Thousand Talents Program (to HY), Break through project of Chinese Academy of Sciences, Shanghai City Committee of science and technology project (16JC1420202 to HY), the Ministry of Science and Technology of China (MOST; 2016YFA0100500).

All procedures including animal subjects have been approved by the Biomedical Research Ethics Committee at the Shanghai Institutes for Biological Science (CAS).
1. Design of Donor Plasmids
<ol>
	<li>Selection of sgRNA
	<ol>
		<li>Use online CRISPR design tools to predict sgRNAs on the target region11,12,13,14,15. ...

<ol>
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The most critical steps in the construction of HMEJ donor plasmids are: (1) selection of the sgRNA with high DNA cleavage efficiency and low distance between sgRNA cutting site and stop codon, and (2) proper construction of HMEJ donor. CRISPR/Cas9-mediated cleavage on both transgene donor vector (containing sgRNA target sites and ~800 bp homology arms) and targeted genome is necessary for efficient and precise targeted integration in vivo. The most critical steps of generation of knock-in mice using the HMEJ-bas...

Precise, targeted genome editing is often required for producing genetically modified animal models and clinical therapies. Much effort has been made to develop various strategies for efficient targeted genome editing, such as zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 systems. These strategies create targeted DNA double-strand breaks (DSB) in the genome, and take advantage of intrinsic DNA repair systems, such as homologous recombination (HR)1,2, microhomology-mediated end joining (MMEJ)3,4,5, and non-homologous end joining (NHEJ)6,7,8 to induce targeted integration of transgenes1,9. The HR-based strategy is currently the most commonly used genome editing approach, which is very efficient in cell lines, but not readily accessible to non-dividing cells due to its restricted occurrence in the late S/G2 phase. Thus, the HR-based strategy is not applicable for in vivo genome editing. Recently, the NHEJ-based strategy was developed for efficient gene knock-in in mouse tissues8. Nevertheless, the NHEJ-based method usually introduces indels at the junctions, making it difficult to generate precise genome editing, especially when trying to construct in-frame fusion genes8. MMEJ-based targeted integration is capable of precise genome editing. However, it only modestly increases the targeted integration efficiency in previous reports5. Therefore, improving the efficiency of precise targeted integration in vivo is urgently needed for broad therapeutic applications3.
In a recently published work, we demonstrated a homology-mediated end joining (HMEJ)-based strategy, which showed the highest targeted integration efficiency in all reported strategies both in vitro and in vivo10. Here, we describe a protocol for the establishment of the HMEJ system, and also the construction of the single-guide RNA (sgRNA) vectors targeting the gene of interest and the donor vectors harboring sgRNA target sites and ~800 bp of homology arms (Figure 1). In this protocol, we also describe the detailed steps for generation of DNA knock-in mice and brief steps for targeted integration in tissues in vivo. Moreover, a proof-of-concept study of the HMEJ-based strategy demonstrated its ability to correct Fah mutation and rescue Fah-/- liver failure mice, which further revealed its therapeutic potential.

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crispr/cas9-mediated targeted integration in vivo using a homology-mediated end joining-based strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

HMEJ-based genome editing in mouse embryos: To define the knock-in efficiency of the HMEJ-based method in mouse zygotes, we delivered Cas9 mRNA, sgRNA targeting the Cdx2 gene and the HMEJ donor into mouse zygotes, which was designed to fuse a p2A-mCherry reporter gene to the last codon of the Cdx2 gene (Figure 2A). The injected zygotes developed into blastocysts in the culture. To evaluate the knock-in effic...

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CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Research

JoVE Journal

Genetics

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

科研

遗传学

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...