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...

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 border="0" cellpadding="0" cellspacing="0" style="width:1205px;" width="1206">
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		<col />
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	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">pX330</td>
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			<td style="width:131px;">42230</td>
			<td style="width:524px;"></td>
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			<td height="31" style="height:31px;width:359px;">pAAV vector</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">37083</td>
			<td style="width:524px;"></td>
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		<tr height="33">
			<td height="33" style="height:33px;width:359px;">pX260</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">42229</td>
			<td style="width:524px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:359px;">AAV_Efs_hSpCas9_NLS_FLAG-SV40</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">97307</td>
			<td style="width:524px;">AAV vector for encoding a human codon-optimized SpCas9 driven by EFs promoter</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:359px;">AAV_Actb HMEJ donor_U6_sgRNA_EF1a_GFP_polyA</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">97308</td>
			<td style="width:524px;">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 height="36">
			<td height="36" style="height:36px;width:359px;">AAV_Cdx2 HMEJ donor</td>
			<td style="width:192px;">Addgene</td>
			<td style="width:131px;">97319</td>
			<td style="width:524px;">HMEJ donor for fusing a p2A-mCherry reporter to mouse Cdx2.&#160;</td>
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			<td style="width:131px;">AM9930</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Bbs I</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">R0539S</td>
			<td style="width:524px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:359px;">NEB Buffer 2</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">B7002S</td>
			<td style="width:524px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:359px;">T7 endonuclease I</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">M0302L</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">NEBuilder HiFi DNA Assembly Master Mix</td>
			<td style="width:192px;">New England Biolabs</td>
			<td style="width:131px;">E2621L</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Plasmid EndoFree-Midi Kit</td>
			<td style="width:192px;">Qiagen</td>
			<td style="width:131px;">12143</td>
			<td style="width:524px;"></td>
		</tr>
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			<td height="40" style="height:40px;width:359px;">MMESSAGE MMACHINE T7 ULTRA</td>
			<td style="width:192px;">Life Technologies</td>
			<td style="width:131px;">AM1345</td>
			<td style="width:524px;"></td>
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			<td style="width:131px;">AM1908</td>
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			<td style="width:192px;">Life Technologies</td>
			<td style="width:131px;">AM1354</td>
			<td style="width:524px;"></td>
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			<td height="40" style="height:40px;width:359px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:192px;">Sutter Instrument</td>
			<td style="width:131px;">P-97</td>
			<td style="width:524px;">&#160;Micropipette Puller (parameters: heat, 74; pull, 60; velocity, 80; time/delay, 200; pressure, 300)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Borosilicate glass</td>
			<td style="width:192px;">Sutter Instrument</td>
			<td style="width:131px;">B100-78-10</td>
			<td style="width:524px;">type of capillaries (outer diameter 1.0 mm, inner diameter 0.78 mm with filament)&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">FemtoJet microinjector</td>
			<td style="width:192px;">Eppendorf</td>
			<td style="width:131px;"></td>
			<td></td>
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			<td height="40" style="height:40px;width:359px;">Freezing microtome</td>
			<td style="width:192px;">Leica</td>
			<td style="width:131px;">CM1950-Cryostat</td>
			<td style="width:524px;">thickness&#160;of&#160;40&#160;&#956;m&#160;for&#160;brain,&#160;10 &#956;m&#160;for&#160;liver</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Rabbit anti-mCherry</td>
			<td style="width:192px;">GeneTex</td>
			<td style="width:131px;"></td>
			<td style="width:524px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Cy3-AffiniPure Goat Anti-Rabbit IgG</td>
			<td style="width:192px;">Jackson Immunoresearch</td>
			<td style="width:131px;"></td>
			<td style="width:524px;"></td>
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			<td height="40" style="height:40px;width:359px;">DMEM</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">11965092</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:359px;">FBS</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">10099141</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:359px;">NEAA</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">11140050</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:359px;">Pen,Strep,Glutamine</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:131px;">10378016</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:359px;">Gel Extraction Kit</td>
			<td style="width:192px;">Omega</td>
			<td style="width:131px;">D2500-02</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">FACS</td>
			<td>BD AriaII</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMSG</td>
			<td>Ningbo Sansheng Medicine</td>
			<td>S141004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCG</td>
			<td>Ningbo Sansheng Medicine</td>
			<td>B141002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytochalasin B</td>
			<td>Sigma</td>
			<td>CAT#C6762</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KSOM+AA with D-Glucose and Phenol Red</td>
			<td>Millipore</td>
			<td>CAT#MR-106-D</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M2 Medium with Phenol Red</td>
			<td>Millipore</td>
			<td>CAT#MR-015-D</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral oil</td>
			<td>Sigma</td>
			<td style="width:131px;"></td>
			<td style="width:524px;"></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...

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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 ...

The overall goal of this HMEJ strategy is to provide a promising genetic tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies. This HMEJ passed the measure. Can have also key questions in the genetic and in neuro field.Such as how to improve the efficiency of precise targeted integration of transgenes in vivo. The main advantage of this HMEJ based strategy is ability to correct gene mutations with high efficiency in vivo. Which followed is therapeutic in potential.Demonstrating the procedure will be Xing Wang, a graduate student from my laboratory. To begin the protocol, collect 5000 N2a cells, previously transfected with Cas9 sgRNA EGFP expression vectors by fluorescence activated cell sorting or FACS, using non-transfected cells as a control. Digest the collected cells in 2 to 5 microliters of lysis buffer at 56 degrees Celsius for 30 minutes.Then heat and activate the proteinase K at 95 degrees Celsius for 10 minutes. Amplify the sample by nested PCR using the manufacturer's protocol. Mix one microliter of the lysis product with DNA polymerase and a pair of outer primers recognizing the sequence around the sgRNA target site and perform the primary PCR in a total volume of 20 microliters.Next, activate DNA polymerase at 95 degrees Celsius for 5 minutes, then perform the primary PCR with a final extension at 72 degrees Celsius for 5 minutes. Perform the secondary PCR using one microliter of primary PCR product and a pair of nested inner primers. Denature and re-anneal 300-600 nanograms of purified PCR product in 20 microliters of 1x TC7EI reaction buffer.Add one microliter of T7EI enzyme to the annealed PCR products and digest them at 37 degrees Celsius for two hours. Then run the digestion product on 2%agarose gel and 1x TAE buffer at 120 volts for 40 minutes, until the fragments are separated. Use ImageJ to determine the band intensities of cut and uncut DNA and calculate the indel frequency.For Cas9 mRNA preparation, add the T7 promoter sequence to the Cas9 coding region by PCR amplification. Next, add the primer Cas9 FR at a final concentration of 0.1 micromolar and 20 nanograms of Cas9 expressing vector to the 1x high fidelity DNA polymerase mix. Adjust the final volume to 50 microliters with nuclease free water.Activate DNA polymerase at 95 degrees Celsius for 5 minutes and perform the PCR. Purify the T7 Cas9 and PCR product for in vitro transcription, or IVT. Then transcribe 0.5 to 1 microgram of DNA, using an mRNA transcription kit at 37 degrees Celsius for 8 hours in a total volume of 20 microliters.Add 1 microliter of DNase to the mixture to remove the DNA template at 37 degrees Celsius for 15 minutes. After adding a poly(A)tail for 45 minutes at 37 degrees Celsius, recover the Cas9 mRNA with an RNA purification kit. Generate the sgRNA template driven by a T7 promoter with high fidelity DNA polymerase as performed in the previous section and choose an sgRNA scaffold containing vector as the template.After purifying the T7 sgRNA PCR product, use 0.5 to 1 microgram of DNA as the template for in vitro transcription of sgRNA using a short RNA transcription kit at 37 degrees Celsius for 6 hours, in a total volume of 20 microliters. After 6 hours of incubation, add 1 microliter of DNase to the mixture and continue the incubation at 37 degrees Celsius for 15 minutes to remove the DNA template. Purify sgRNAs again using the RNA purification kit.Dilute the sgRNA to 500 nanograms per microliter in RNase free water and store the samples at minus 80 degrees Celsius for up to three months. High quality of Cas9 mRNA and sgRNA with no degeneration and the correct line of HMEJ donor parameters are the most critical steps in the generation of knock-in mice. Place the fertilized embryos into KSOM medium at 37 degrees Celsius in an incubator with 5%carbon dioxide.Mix 100 nanograms per microliter of Cas9 mRNA, 50 nanograms per microliter of sgRNA, and 100 nanograms per microliter of HMEJ donor vector and add water to adjust the final volume to 10 microliters. Pipette the mixture up and down and place it on ice. Next, pull capillary needles using a micropipette puller.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 micrograms per milliliter Cytochalasin B using a microinjector with constant flow settings. Finally, culture the injected zygotes in KSOM medium at 37 degrees Celsius under 5%carbon dioxide until the blastocyst stage after 3 1/2 days for fluorescence observation. After 3 days of injections, 12.9%of the blastocysts receiving HMEJ donors were positive for mCherry, which was strictly expressed in the trophectoderm.By sequencing the PCR positive mice, all examined integration events were precise in-frame integrations at both 5 prime and 3 prime junctions. After 7 days of injections, immunofluorescent images revealed that nearly half of the transfected hepatocytes expressed mCherry as stained on the liver sections. After the withdrawal of NTBC, immunohistochemistry liver staining of Fah corrected hepatocytes of Fah negative mice receiving Fah HMEJ and Cas9 constructs showed more effective proliferation than MMEJ based method.The HMEJ based strategy could be an ideal platform for replacing mutated sequence such as part of mutation with the correct one. Which is not applicable for an HHEJ based method. After this experiment, this therapy paves the way for researchers into field where precise target integration to explore broad therapy applications.

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14.6K 次观看.  Chinese Academy of Sciences. The overall goal of this HMEJ strategy is to provide a promising genetic tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies. This HMEJ passed the measure. Can have also key questions in the genetic and in neuro field.Such as how to improve the efficiency of precise targeted integration of transgenes in vivo. The main advantage of this HMEJ based strategy is ability to correct gene mutations with high efficiency in ...