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. 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.</li>
		<li>Linearize 2 &#181;g of Cas9-CMV-EGFP expression vectors and sgRNA by BbsI digestion (1 &#181;L of BbsI for 2 h at 37 &#176;C at a final concentration of 1 U/&#181;L in a volume of 20 &#181;L). Then purify the product by gel purification kit with a 1% agarose gel in 1&#215; TAE buffer.</li>
		<li>Mix a pair of sgRNA oligonucleotides in 10 &#181;L of 1&#215; T4 DNA ligase buffer to a final concentration of 50 &#181;M. Incubate the oligo solution using a temperature gradient from 95 &#176;C to 25 &#176;C with a temperature change rate of 5 &#176;C /5 min (95 &#176;C for 5 min, then 90 &#176;C for 5 min, 85 &#176;C for 5 min, etc.), which will anneal the oligos.</li>
		<li>Mix 4 &#181;L of annealed product, 2 &#181;L of the linearized vector with 1 &#181;L of T4 DNA ligase in 10 &#181;L of 1&#215; T4 DNA ligase buffer, and then ligate at 22 &#176;C for 1-2 h (Figure 1B).</li>
	</ol>
	</li>
	<li>T7 endonuclease assay of sgRNA 
	NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI)&#160;assay17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon.
	<ol>
		<li>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 &#176;C in 5% CO2.</li>
		<li>After 48 h of incubation, collect 5,000 transfected cells (GFP+) by fluorescence-activated cell sorting (FACS) using non-transfected cells as a control.</li>
		<li>Digest the collected cells in 2-5 &#181;L oflysis buffer (0.1% Triton X-100, 0.1% Tween 20, and 100 &#181;g/mL Proteinase K) at 56 &#176;C for 30 min, and then heat inactivate proteinase K at 95 &#176;C for 10 min.</li>
		<li>Amplify the sample by nested PCR (Table 1) using the manufacturer&#39;s protocol. The size of PCR products is set to 300-500 bp.
		<ol>
			<li>Mix 1 &#181;L of lysis product with DNA polymerase and a pair of outer primers recognizing the sequence around the sgRNA target site (0.1 &#181;M, final concentration) (Table 1), and perform the primary PCR in a volume of 20 &#181;L.</li>
			<li>Activate DNA polymerase at 95 &#176;C for 5 min, and perform the primary PCR for 30 cycles at 95 &#176;C for 30 s, 60 &#176;C for 30 s, and 72 &#176;C for 24 s (1 min/1 kb), with a final extension at 72 &#176;C for 5 min.</li>
			<li>Perform the secondary PCR using 1 &#181;L of primary PCR product and a pair of nested inner primers.</li>
		</ol>
		</li>
		<li>Denature and re-anneal 300-600 ng of purified PCR product in 20 &#181;L of 1&#215; 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 &#176;C to 25 &#176;C with a rate of 5 &#176;C /5 min.</li>
		<li>Add 1 &#181;L of T7EI enzyme to the annealed PCR products and digest at 37 &#176;C for 2 h. Then run the digestion product on 2% agarose gel in 1&#215;TAE buffer at 120 V for 40 min until the fragments are separated (see Table of Materials).</li>
		<li>Use ImageJ to determine the band intensities of cut and uncut DNA. Calculate the indel frequency using the methods as previously reported9 (Figure 1C).</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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 &#181;M final concentration using mouse genomic DNA at 200 ng/&#181;L (Figure 1D, Table 1).</li>
		<li>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 &#181;M final concentration using genomic DNA or plasmid with reporter sequences at 100 ng/&#181;L or 30 ng/&#181;L (Figure 1D, Table 1).</li>
		<li>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 &#181;M final concentration using mouse genomic DNA at 200 ng/&#181;L (Figure 1D, Table 1).</li>
		<li>Run all the PCR products on 1% agarose gel in 1&#215;TAE buffer, and purify the PCR products of expected size by gel extraction kit, according to the manufacturer&#39;s instructions (Table 1).</li>
		<li>Digest 50-100 ng of a construct vector with by KpnI and XbaI. Mix 2 &#181;L of the linearized vector at 30-40 ng/&#181;L with three PCR amplified fragments (1 &#181;L for each, 100-200 ng/&#181;L) in 2x Gibson mix. Add H2O to adjust the final volume to 10 &#181;L.Incubate the mix at 50 &#176;C for 60 min.</li>
		<li>Transform competent E. coli cells with all the assembled product and extract the plasmid constructs by DNA extraction kit according to the manufacturer&#39;s instructions. Verify the HMEJ donor by DNA sequencing.</li>
	</ol>
	</li>
</ol>
2. Genome Editing in Mouse Embryos Using the HMEJ-Based Method
<ol>
	<li>Production of Cas9 mRNA
	<ol>
		<li>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 &#181;M and 20 ng of Cas9 expressing vector to 1&#215; high fidelity DNA polymerase mix. Adjust the final volume to 50 &#181;L with H2O.</li>
		<li>Activate DNA polymerase at 95 &#176;C for 5 min, and perform the PCR for 36 cycles at 95 &#176;C for 30 s, 60 &#176;C for 30 s, and 68&#160;&#176;C for 4 min (1 min/1 kb), with a final extension at 68&#160;&#176;C for 10 min.</li>
		<li>Purify T7-Cas9 PCR product for in vitro transcription (IVT), and then transcribe 0.5-1 &#181;g DNA by mRNA transcription kit at 37 &#176;C for 8 h in a total volume of 20 &#181;L, according to the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Add 1 &#181;L of DNase to the mixture to remove the DNA template at 37 &#176;C for 15 min. Add a poly-A tail for 45 min at 37 &#176;C and recover the Cas9 mRNA by RNA purification kit, according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	</ol>
	</li>
	<li>Production of sgRNA
	<ol>
		<li>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.</li>
		<li>Purify the T7-sgRNA PCR product and use 0.5-1 &#181;g DNA as the template for in vitro transcription of sgRNA using a short RNA transcription kit at 37 &#176;C for 6 h in a total volume of 20 &#181;L, according to the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Add 1 &#181;L of DNase to the mixture and continue the incubation at 37 &#176;C for 15 min to remove the DNA template. Purify the sgRNAs by RNA purification kit, as above (see Table of Materials).</li>
		<li>Dilute the sgRNA to 500 ng/&#181;L in RNase-free water and store the samples at &#8722;80 &#176;C for up to 3 months. 
		NOTE: CRISPR ribonucleoproteins (RNPs) are an alternative substitution with a better cutting efficiency18,19,20.</li>
	</ol>
	</li>
	<li>Embryo collection, microinjection and in vitro culture
	<ol>
		<li>Superovulate female B6D2F1 (C57BL/6 &#215; 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.</li>
		<li>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.</li>
		<li>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&#8226;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&#8226;2H2O, 0.004 g/L EDTA, 0.146 g/L L-glutamine, and 1 g/L Bovine serum albumin) at 37 &#176;C in an incubator with 5% CO2.</li>
		<li>Mix Cas9 mRNA (100 ng/&#181;L), sgRNA (50 ng/&#181;L), and HMEJ donor vector (100 ng/&#181;L), and add H2O to adjust the final volume to 10 &#181;L. Put the mixture on ice.</li>
		<li>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.</li>
		<li>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 &#181;g/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.</li>
		<li>Culture the injected zygotes in KSOM medium at 37 &#176;C under 5% CO2 until the blastocyst stage after 3.5 days for fluorescence observation (Figures 2B and 2C).</li>
	</ol>
	</li>
	<li>Embryo transfer and generation of mice
	<ol>
		<li>Mate estrous ICR female mice with vasectomized ICR male mice on the same day as injection.</li>
		<li>Culture the injected zygotes into the 2-cell stage at 37 &#176;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.</li>
	</ol>
	</li>
	<li>Mouse genotyping
	<ol>
		<li>Extract mouse genomic DNA from toe or tail samples using a DNA extraction kit, according to the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Identify the 5&#39; and 3&#39; junction of knock-in events using 200-400 ng of genomic DNA measured by UV/vis spectrometry as a template to perform the PCR ampli&#64257;cation.
		<ol>
			<li>Activate DNA polymerase at 95 &#176;C for 5 min, and perform the PCR for 38&#160;cycles at 95 &#176;C for 30 s, 60 &#176;C for 30 s, and 72 &#176;C for 1&#160;min (1 min/1 kb), with a final extension at 72 &#176;C for 10 min. For the 5&#39; 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&#39; junction, use the forward primer on the knock-in fragment (p2A-mCherry), with the reverse one on the downstream of the HAR (Table 1).</li>
		</ol>
		</li>
		<li>Run 6 &#181;L of the PCR product on 1% agarose gel in 1&#215;TAE buffer and check for the expected fragment size. Then verify them by DNA sequencing (Figure 2D).</li>
	</ol>
	</li>
</ol>
3. HMEJ-Based In Vivo Genome Editing in Hepatocytes
<ol>
	<li>Place recipient C57BL/6J mouse (8 week) in a restraining device and put the tail through the slit.</li>
	<li>Mix HMEJ donor vectors (30 &#181;g) and spCas9 expression vectors (30 &#181;g) in 2 mL of saline solution. For the control experiment, suspend HMEJ donor vectors (30 &#181;g) in 2 mL of saline solution (Figure 3A).</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Dehydrate the tissue using 30% sucrose overnight, until it sinks to the bottom of tube.</li>
	<li>Section the frozen tissue at a thickness of 10 &#181;m for liver samples.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Counterstain the sections with DAPI for 20 min and mount with glycerin on glass slides for further fluorescence observation (Figure 3B).</li>
</ol>

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The authors have nothing to disclose.

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...

An erratum was issued for: <a href="https://www.jove.com/t/56844/crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated">Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells</a>. The phrases&#160;&#34;surveyor assay&#34;&#160;and&#160;&#34;Surveyor&#160;Nuclease&#34; have&#160;been updated to &#34;T7E1 assay&#34;&#160; to &#34; T7&#160;endonuclease I&#34; respectively.&#160;
Step 1.2 in&#160;the Protocol has been updated from:
<ol class="jove_content" start="2">
	<li>Surveyor nuclease&#160;assay of sgRNA 
	NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by surveyor nuclease assay (also known as T7 endonuclease I (T7EI) assay)17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon.</li>
</ol>
to:
<ol class="jove_content" start="2">
	<li>T7 endonuclease assay of sgRNA 
	NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI)&#160;assay17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon.</li>
</ol>
Figure 1 in the Representative Results has been updated&#160;from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56844/56844fig1.jpg" /> 
Figure 1: HMEJ-mediated targeted integration in vitro. 
(A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2a&#160;cells. GFP+ cells were sorted at day 3 for surveyor assay. (C) Surveyor assay for Cdx2 targeting: 6 different sgRNAs were designed for surveyor assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.&#160;<a href="https://cloudfront.jove.com/files/ftp_upload/56844/56844fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56844/56844fig1v3.jpg" /> 
Figure 1: HMEJ-mediated targeted integration in vitro. 
(A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2a&#160;cells. GFP+ cells were sorted at day 3 for T7EI&#160;assay. (C) T7EI assay for Cdx2 targeting: 6 different sgRNAs were designed for T7EI assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.&#160;<a href="https://cloudfront.jove.com/files/ftp_upload/56844/56844fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for: <a href="https://www.jove.com/t/56844/crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated">Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells</a>. The phrases&#160;&#34;surveyor assay&#34;&#160;and&#160;&#34;Surveyor&#160;Nuclease&#34; have&#160;been updated to &#34;T7E1 assay&#34;&#160; to &#34; T7&#160;endonuclease I&#34; respectively.&#160;

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

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.

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

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...

Watch this Scientific Journal Video about CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy at JoVE.com

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 ...

crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated

Research

JoVE Journal

Genetics

14.7K Views.  Chinese Academy of Sciences. De geclusterde regelmatig interspaced korte palindromische herhalingen/CRISPR geassocieerde eiwit 9 (CRISPR/Cas9) systeem biedt een veelbelovend instrument voor de gentechnologie, en opent de mogelijkheid van gerichte integratie van transgenen. We beschrijven een homologie-gemedieerde einde deelname aan (HMEJ)-gebaseerd strategie voor efficiënte DNA integratie in vivo gerichte en gerichte gentherapieën met behulp van CRISPR/Cas9.

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

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

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

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

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Detecting Off-Target CRISPR-Cas9 Cleavage Events with CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

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

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