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)3,4,5, 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.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by CAS Strategic Priority Research Program (XDB02050007, XDA01010409), the National Hightech R&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).
Materials
| 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 |
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