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 transgenes^1,9. 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

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 potential off-targets (Figure 1A)¹⁶.
   2. Linearize 2 µg of Cas9-CMV-EGFP expression vectors and sgRNA by BbsI digestion (1 µL of BbsI for 2 h at 37 °C at a final concentration of 1 U/µL in a volume of 20 µL). Then purify the product by gel purification kit with a 1% agarose gel in 1× TAE buffer.
   3. Mix a pair of sgRNA oligonucleotides in 10 µL of 1× T4 DNA ligase buffer to a final concentration of 50 µM. Incubate the oligo solution using a temperature gradient from 95 °C to 25 °C with a temperature change rate of 5 °C /5 min (95 °C for 5 min, then 90 °C for 5 min, 85 °C for 5 min, etc.), which will anneal the oligos.
   4. Mix 4 µL of annealed product, 2 µL of the linearized vector with 1 µL of T4 DNA ligase in 10 µL of 1× T4 DNA ligase buffer, and then ligate at 22 °C for 1-2 h (Figure 1B).
2. T7 endonuclease assay of sgRNA
   NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI) assay¹⁷. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon.
   1. Transfect Cas9-sgRNA-EGFP expression vectors into N2a cell lines cultured in DMEM supplemented with 10% fetal bovine serum, 1% PSG, and 1% non-essential amino acid by the transfection kit (see Table of Materials). Incubate the transfected cells at 37 °C in 5% CO₂.
   2. After 48 h of incubation, collect 5,000 transfected cells (GFP⁺) by fluorescence-activated cell sorting (FACS) using non-transfected cells as a control.
   3. Digest the collected cells in 2-5 µL oflysis buffer (0.1% Triton X-100, 0.1% Tween 20, and 100 µg/mL Proteinase K) at 56 °C for 30 min, and then heat inactivate proteinase K at 95 °C for 10 min.
   4. Amplify the sample by nested PCR (Table 1) using the manufacturer's protocol. The size of PCR products is set to 300-500 bp.
      1. Mix 1 µL of lysis product with DNA polymerase and a pair of outer primers recognizing the sequence around the sgRNA target site (0.1 µM, final concentration) (Table 1), and perform the primary PCR in a volume of 20 µL.
      2. Activate DNA polymerase at 95 °C for 5 min, and perform the primary PCR for 30 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 24 s (1 min/1 kb), with a final extension at 72 °C for 5 min.
      3. Perform the secondary PCR using 1 µL of primary PCR product and a pair of nested inner primers.
   5. Denature and re-anneal 300-600 ng of purified PCR product in 20 µL of 1× T7EI reaction buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT pH 7.9) using a gradient of temperature from 95 °C to 25 °C with a rate of 5 °C /5 min.
   6. Add 1 µL of T7EI enzyme to the annealed PCR products and digest at 37 °C for 2 h. Then run the digestion product on 2% agarose gel in 1×TAE buffer at 120 V for 40 min until the fragments are separated (see Table of Materials).
   7. Use ImageJ to determine the band intensities of cut and uncut DNA. Calculate the indel frequency using the methods as previously reported⁹ (Figure 1C).
3. Construction of donor vector
   NOTE: To generate HMEJ donor vectors for Cdx2 gene, construct a donor DNA (800 bp HAL-p2A-mCherry-800 bp HAR) flanked with 23 nt Cdx2-sgRNAs targeting sequence at both ends (Figure 1D and Figure 1E). The PAM of target sequence was adjacent to the end of the homologous arm. Gibson assembly is recommended for HMEJ donor cloning.
   1. Amplify the 800 bp left homology arm (HAL) with forward primer-1F (containing 15-20 nt overlap sequence from vector, 23 nt Cdx2-sgRNA targeting sequence, and about 20 nt sequence from HAL) and reverse primer-1R (containing 15-20 bp overlap sequence from p2A-mCherry and about 20 nt sequence from HAL) at 0.1 µM final concentration using mouse genomic DNA at 200 ng/µL (Figure 1D, Table 1).
   2. Amplify the p2A-mCherry insertion fragment with forward primer-2F (containing 15-20 nt overlap sequence from HAL and about 20 nt sequence from insertion fragment) and reverse primer-2R (containing 15-20 nt overlap sequence from HAR and about 20 nt sequence from the insertion fragment) at 0.1 µM final concentration using genomic DNA or plasmid with reporter sequences at 100 ng/µL or 30 ng/µL (Figure 1D, Table 1).
   3. Amplify the 800 bp right homology arm (HAR) with forward primer-3F (containing 15-20 nt overlap sequence from vector, 23 nt Cdx2-sgRNA targeting sequence, and about 20 nt sequence from HAR) and reverse primer-3R (containing 15-20 nt overlap sequence from p2A-mCherry and about 20 nt sequence from HAR) at 0.1 µM final concentration using mouse genomic DNA at 200 ng/µL (Figure 1D, Table 1).
   4. Run all the PCR products on 1% agarose gel in 1×TAE buffer, and purify the PCR products of expected size by gel extraction kit, according to the manufacturer's instructions (Table 1).
   5. Digest 50-100 ng of a construct vector with by KpnI and XbaI. Mix 2 µL of the linearized vector at 30-40 ng/µL with three PCR amplified fragments (1 µL for each, 100-200 ng/µL) in 2x Gibson mix. Add H₂O to adjust the final volume to 10 µL.Incubate the mix at 50 °C for 60 min.
   6. Transform competent E. coli cells with all the assembled product and extract the plasmid constructs by DNA extraction kit according to the manufacturer's instructions. Verify the HMEJ donor by DNA sequencing.

2. Genome Editing in Mouse Embryos Using the HMEJ-Based Method

1. Production of Cas9 mRNA
   1. For Cas9 mRNA preparation, add the T7 promoter sequence to the Cas9 coding region by PCR amplification using the appropriate primer pair listed in Table 1. Add the primer Cas9 F/R at a final concentration of 0.1 µM and 20 ng of Cas9 expressing vector to 1× high fidelity DNA polymerase mix. Adjust the final volume to 50 µL with H₂O.
   2. Activate DNA polymerase at 95 °C for 5 min, and perform the PCR for 36 cycles at 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 4 min (1 min/1 kb), with a final extension at 68 °C for 10 min.
   3. Purify T7-Cas9 PCR product for in vitro transcription (IVT), and then transcribe 0.5-1 µg DNA by mRNA transcription kit at 37 °C for 8 h in a total volume of 20 µL, according to the manufacturer's instructions (see Table of Materials).
   4. Add 1 µL of DNase to the mixture to remove the DNA template at 37 °C for 15 min. Add a poly-A tail for 45 min at 37 °C and recover the Cas9 mRNA by RNA purification kit, according to the manufacturer's instructions (see Table of Materials).
2. Production of sgRNA
   1. Generate the sgRNA template driven by a T7 promoter with high fidelity DNA polymerase as above. Choose an sgRNA scaffold containing vector as the template. The primers used are listed in Table 1.
   2. Purify the T7-sgRNA PCR product and use 0.5-1 µg DNA as the template for in vitro transcription of sgRNA using a short RNA transcription kit at 37 °C for 6 h in a total volume of 20 µL, according to the manufacturer's instructions (see Table of Materials).
   3. Add 1 µL of DNase to the mixture and continue the incubation at 37 °C for 15 min to remove the DNA template. Purify the sgRNAs by RNA purification kit, as above (see Table of Materials).
   4. Dilute the sgRNA to 500 ng/µL in RNase-free water and store the samples at −80 °C for up to 3 months.
      NOTE: CRISPR ribonucleoproteins (RNPs) are an alternative substitution with a better cutting efficiency^18,19,20.
3. Embryo collection, microinjection and in vitro culture
   1. Superovulate female B6D2F1 (C57BL/6 × DBA2J) mice (7-8 weeks old) by pregnant mare serum gonadotropin (PMSG), followed by human chorionic gonadotropin (hCG) 48 h later. After the hCG injection, house females with B6D2F1 males overnight.
   2. Sacrifice the females by CO₂ anesthesia, 24 h after hCG injection. Collect the fertilized embryos from their oviducts (with 30 - 50 embryos for each female) in M2 medium.
   3. Place the fertilized embryos (about 300 eggs for one-day injection) into KSOM medium (5.55 g/L NaCl, 0.19 g/L KCl, 0.05 g/L KH₂PO₄, 0.05 g/L MgSO₄•7H₂O, 0.04 g/L Glucose, 1.12 g/L Sodium lactate, 2.1 g/L NaHCO₃, 0.02 g/L Sodium pyruvate, 0.25 g/L CaCl₂•2H₂O, 0.004 g/L EDTA, 0.146 g/L L-glutamine, and 1 g/L Bovine serum albumin) at 37 °C in an incubator with 5% CO₂.
   4. Mix Cas9 mRNA (100 ng/µL), sgRNA (50 ng/µL), and HMEJ donor vector (100 ng/µL), and add H₂O to adjust the final volume to 10 µL. Put the mixture on ice.
   5. Pull capillary needles (outer diameter 1.0 mm, inner diameter 0.78 mm with filament) using a Micropipette Puller (parameters: heat, 74; pull, 60; velocity, 80; time/delay, 200; pressure, 300. See Table of Materials). Commercial needles would be an alternative substitution for the microinjection.
   6. Inject a probable volume of the mixture into the cytoplasm of zygotes with well-defined pronuclei in a droplet of HEPES-CZB medium containing 5 µg/mL cytochalasin B using a microinjector with constant flow settings (Figure 2A) (see Table of Materials)²¹.
      NOTE: Each group of zygotes should be injected within 20-30 min. Cytochalasin B could increase the viability of mouse zygote after injection. Alternatively, microinjection can be operated with the piezo system, as described previously²².
   7. Culture the injected zygotes in KSOM medium at 37 °C under 5% CO₂ until the blastocyst stage after 3.5 days for fluorescence observation (Figures 2B and 2C).
4. Embryo transfer and generation of mice
   1. Mate estrous ICR female mice with vasectomized ICR male mice on the same day as injection.
   2. Culture the injected zygotes into the 2-cell stage at 37 °C under 5% CO₂, and transfer 25-30 2-cell embryos into oviducts of pseudopregnant ICR females at 0.5 day post coitum (dpc). Recipient mothers deliver pups at 19.5 dpc.
5. Mouse genotyping
   1. Extract mouse genomic DNA from toe or tail samples using a DNA extraction kit, according to the manufacturer's instructions (see Table of Materials).
   2. Identify the 5' and 3' junction of knock-in events using 200-400 ng of genomic DNA measured by UV/vis spectrometry as a template to perform the PCR amplification.
      1. Activate DNA polymerase at 95 °C for 5 min, and perform the PCR for 38 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min (1 min/1 kb), with a final extension at 72 °C for 10 min. For the 5' junction, use the forward primer at the upstream of the HAL, with the reverse one on the knock-in fragment (p2A-mCherry). As to 3' junction, use the forward primer on the knock-in fragment (p2A-mCherry), with the reverse one on the downstream of the HAR (Table 1).
   3. Run 6 µL of the PCR product on 1% agarose gel in 1×TAE buffer and check for the expected fragment size. Then verify them by DNA sequencing (Figure 2D).

3. HMEJ-Based In Vivo Genome Editing in Hepatocytes

1. Place recipient C57BL/6J mouse (8 week) in a restraining device and put the tail through the slit.
2. Mix HMEJ donor vectors (30 µg) and spCas9 expression vectors (30 µg) in 2 mL of saline solution. For the control experiment, suspend HMEJ donor vectors (30 µg) in 2 mL of saline solution (Figure 3A).
3. Clean the mouse tail with 70% ethanol. Insert the needle into the tail vein and inject the plasmid DNA solution within 5-7 s. Remove the needle and release the mouse from the restraining device.
4. Sacrifice mice by CO₂ anesthesia after 5-9 days after injection. Perfuse the mice transcardially with 0.9% saline, followed by 4% paraformaldehyde using a peristaltic pump, and fix the liver overnight at 4 °C.
5. Dehydrate the tissue using 30% sucrose overnight, until it sinks to the bottom of tube.
6. Section the frozen tissue at a thickness of 10 µm for liver samples.
7. Rinse the sections three times in 0.1 M phosphate-buffered (PB) and incubate them with primary antibody: Rabbit anti-mCherry (diluted in 5% NGS) overnight at 4 °C.
8. Wash sections three times in PB, and then incubate them with secondary antibody: Cy3-AffiniPure Goat Anti-Rabbit IgG for 2 h at room temperature on an orbital shaker.
9. Counterstain the sections with DAPI for 20 min and mount with glycerin on glass slides for further fluorescence observation (Figure 3B).

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