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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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

1. Selection of sgRNA
   1. Use online CRISPR design tools to predict sgRNAs on the target region^{11,12,13,14,15}. For the Cdx2 locus, design six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon with higher rank and lower ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pX330	Addgene	42230
pAAV vector	Addgene	37083
pX260	Addgene	42229
AAV_Efs_hSpCas9_NLS_FLAG-SV40	Addgene	97307	AAV vector for encoding a human codon-optimized SpCas9 driven by EFs promoter
AAV_Actb HMEJ donor_U6_sgRNA_EF1a_GFP_polyA	Addgene	97308	HMEJ donor for fusing a p2A-mCherry reporter to mouse Actb. EGFP driven by EF1a promoter and U6-driven sgRNAs targeting Actb. AAV backbone.
AAV_Cdx2 HMEJ donor	Addgene	97319	HMEJ donor for fusing a p2A-mCherry reporter to mouse Cdx2.
Lipofectamine 3000 Transfection Reagent	Life Technology	L3000015
Nuclease-Free Water	Life Technologies	AM9930
Bbs I	New England Biolabs	R0539S
NEB Buffer 2	New England Biolabs	B7002S
T7 endonuclease I	New England Biolabs	M0302L
NEBuilder HiFi DNA Assembly Master Mix	New England Biolabs	E2621L
Plasmid EndoFree-Midi Kit	Qiagen	12143
MMESSAGE MMACHINE T7 ULTRA	Life Technologies	AM1345
MEGACLEAR KIT 20 RXNS	Life Technologies	AM1908
MEGASHORTSCRIPT T7 KIT 25 RXNS	Life Technologies	AM1354
Flaming/Brown Micropipette Puller	Sutter Instrument	P-97	Micropipette Puller (parameters: heat, 74; pull, 60; velocity, 80; time/delay, 200; pressure, 300)
Borosilicate glass	Sutter Instrument	B100-78-10	type of capillaries (outer diameter 1.0 mm, inner diameter 0.78 mm with filament)
FemtoJet microinjector	Eppendorf
Freezing microtome	Leica	CM1950-Cryostat	thickness of 40 μm for brain, 10 μm for liver
Rabbit anti-mCherry	GeneTex
Cy3-AffiniPure Goat Anti-Rabbit IgG	Jackson Immunoresearch
DMEM	Gibco	11965092
FBS	Gibco	10099141
NEAA	Gibco	11140050
Pen,Strep,Glutamine	Gibco	10378016
Gel Extraction Kit	Omega	D2500-02
FACS	BD AriaII
PMSG	Ningbo Sansheng Medicine	S141004
HCG	Ningbo Sansheng Medicine	B141002
Cytochalasin B	Sigma	CAT#C6762
KSOM+AA with D-Glucose and Phenol Red	Millipore	CAT#MR-106-D
M2 Medium with Phenol Red	Millipore	CAT#MR-015-D
Mineral oil	Sigma