We thank Prof. Elly M. Tanaka for her continuous and long-term support. This work was supported by a National Natural Science Foundation of China (NSFC) Grant (317716), Research Starting Grants from South China Normal University (S82111 and 8S0109), and a China Postdoctoral Science Foundation Grant (2018M633067).

All animal experiments must be carried out in accordance with local and national regulations on animal experimentation and with approval of the relevant institutional review board.
1. Preparing the CAS9-gRNA RNP mix
<ol>
	<li>Design and synthesize gRNAs. 
	NOTE: Refer to other publications for designing and synthesizing gRNAs, including one exclusively concerning axolotls15,20,21,22.</li>
	<li>Obtain CAS9-NLS protein by preparing in-house or purchasing it commercially.</li>
	<li>Prepare CAS9-gRNA mix by mixing 5 &#181;g of CAS9-NLS protein, 4 &#181;g of gRNA, 0.9 &#181;L of 10x CAS9 buffer, and fill up the volume to 10 &#181;L with nuclease-free H2O. Aliquot the mixture and store at -80 &#176;C if not used immediately.</li>
</ol>
2. Preparing agarose plates for electroporation
<ol>
	<li>Prepare 200 mL of 2% (wt/vol) agarose solution in 1x DPBS. Dissolve by heating the solution in a microwave and pour it onto 10 cm Petri dishes to a depth of about 10 mm (deep enough to cover the whole animal).</li>
	<li>Allow the plates to solidify at room temperature. Store plates at 4 &#176;C if not used immediately.</li>
	<li>Using surgical scalpels, cut the agarose plate to make a slit for holding the tail straight, a well for fitting in the body of the animal, and two extra wells for placing the electrodes (Figure 1E).</li>
	<li>Place the plate on ice and fill it up to the brim with ice-cold 1x DPBS. Use the same DPBS solution for making the plates to ensure consistent electrical conductibility in the set-up.</li>
</ol>
3. Configuring the electroporator
<ol>
	<li>Configure the electroporator. Set up the poring pulse to consist of one bipolar pulse of 70 V, with a duration of 5 ms, interval of 50 ms, and no voltage decay. Set up the transfer pulse to have four bipolar pulses of 40 V, with a duration of 50 ms, interval of 999 ms, and 10% voltage decay.</li>
	<li>Connect the electrodes to the electroporator and adjust the tweezers to be 7 mm apart. Submerge the electrodes in a beaker of DPBS before and in between electroporation.</li>
</ol>
4. Preparing and loading microinjection glass capillaries
<ol>
	<li>Perform a ramp test on a flaming/brown micropipette puller as per the manufacturer&#8217;s instructions to determine the heat value.</li>
	<li>Pull injection capillaries with tapered ends using the following parameters: heat = ramp test value, pull = 100, velocity = 100, time = 100.</li>
	<li>Observe the needle under a stereomicroscope. Using fine forceps, break the capillary at an angle so that it has a slanted tip. Break at a position where it is thin enough to target the central canal, while being strong enough to pierce the skin. 
	NOTE: The point at which the diameter is at about 20 &#181;m or about 50% of the diameter of the spinal cord is a good starting point. Multiple tries may be needed to obtain optimal breaking.</li>
	<li>Add Fast Green FCF solution to the RNP mix at a ratio of 1:30 and load about 5 &#181;L of injection mix into the capillary with a Microloader pipette tip.</li>
	<li>Mount the capillary on the pneumatic pump, with the slanted tip facing downwards.</li>
</ol>
5. Configuring the pneumatic pump
<ol>
	<li>Configure the pneumatic pump. Set the hold to 0.5 pounds per square inch (psi), eject to 2 psi hold, and duration to gated.</li>
	<li>Test capillary and pneumatic pump settings by dipping the capillary into a drop of water and pressing the foot pedal to inject. Ensure that the injection mix comes out in a slow but steady stream.</li>
	<li>Adjust the hold pressure if the injection mix start leaking out spontaneously or water is sucked up the capillary. Increase the ejection pressure or break more of the capillary if the injection mix is coming out too slow. Decrease the ejection pressure if the injection is coming out too fast.</li>
</ol>
6. Injecting RNP mix into the spinal cord
<ol>
	<li>Anesthetize axolotls in 0.03% benzocaine solution for at least 10 min. Check that the animals are not responsive by flipping them upside down in the water.</li>
	<li>Pick up an animal with ring forceps and lay it on a bed of silicon, with the left side of the animal facing up and the tail pointing towards the right. Position the injection site in the middle of the field of view of the stereomicroscope (Figure 1A).</li>
	<li>Adjust the micromanipulator so that the capillary is coming in at 60&#176; from the right side of the field of view (Figure 1B).</li>
	<li>Identify the spinal cord and central canal (Figure 1C). 
	NOTE: The spinal cord is the tubular structure above the notochord.</li>
	<li>Aiming at the central canal, move the capillary forward to gently pierce the skin and muscle into the central canal of the spinal cord. Limit the movement of the capillary to small steps, as the spinal cord is right beneath the tail muscles.</li>
	<li>Press and hold down the foot pedal to inject the mix into the central canal. Observe whether the RNP mix spreads along the central canal as a blue line in both directions, which shows that the capillary has been correctly positioned (Figure 1D).</li>
	<li>If this does not occur, keep the foot pedal pressed while moving the capillary slightly in and out until the RNP mix starts going in. If the capillary becomes clogged by tissue, clear it by ejecting into water at higher pressures.</li>
	<li>Adjust the ejection pressure so that it is just enough to cause the RNP mix to spread in the central canal, but not so much that it blows up the structure.</li>
	<li>Continue to hold down the foot pedal so the injection mix spreads further along the central canal, until it reaches the terminal vesicle at the end of the spinal cord and ventricles in the brain. 
	NOTE: This may take up to 2 min depending on the size of the animal. It may be necessary to increase the pressure after some time to cause the RNP mix to enter the brain ventricles.</li>
	<li>Proceed as soon as possible to electroporation after successful injection.</li>
</ol>
7. Electroporation
<ol>
	<li>Transfer the animal with ring forceps onto the prepared agarose electroporation plate (Figure 1E) on ice. Place the tail inside the slit so it is sandwiched by agarose (Figure 1F).</li>
	<li>Place the electrodes into the wells on both sides of the tail. Ensure that the entire animal and electrodes are covered by ice-cold PBS.</li>
	<li>Press the foot switch to start the electroporation.</li>
	<li>After electroporation is complete, return the animal to water.</li>
	<li>Proceed to inject more animals if necessary.</li>
	<li>Check that the electroporated animals are healthy after anesthesia wears off and that there is no damage done to the tail.</li>
</ol>
8. Assessing knock-out efficiency
<ol>
	<li>Fix the tail of the electroporated animal. Make cross-sections of the tail followed by immunohistochemical staining to assess the efficiency of the knock-out at the protein level. 
	NOTE: If a tail amputation is to be performed after electroporation, the cut off tail could be used for this purpose. Animals electroporated with gRNAs against Gfp or Tyrosinase, for example, can be used as a negative control4.</li>
	<li>Alternatively, the electroporated spinal cord could be extracted and lysed for DNA. Perform genotyping PCR followed by Sanger sequencing or next-generation sequencing to assess the percentage of edited genetic loci15. 
	NOTE: The resulting editing efficiency will underestimate the actual editing efficiency for NSCs, due to the inclusion of non-electroporated cells and neurons that lack apical contact to the central lumen and thus will not receive the RNP mix.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+Central+Nervous+System+During+Salamander+Tail+Regeneration+from+the+Implanted+Neurospheres.">Reconstitution of the Central Nervous System During Salamander Tail Regeneration from the Implanted Neurospheres.</a> Plant, Soil and Environment. 916 (8), 197-202 (2012).</li><li>Nordlander, R. H., Singer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ependyma+in+regeneration+of+the+spinal+cord+in+the+urodele+amphibian+tail.">The role of ependyma in regeneration of the spinal cord in the urodele amphibian tail.</a> Journal of Comparative Neurology. 180 (2), 349-373 (1978).</li><li>Fei, J. 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The authors declare no conflicts of interest.

The described protocol allows time and space restricted gene knock-out in the NSCs in the axolotl spinal cord. The current protocol allows specific targeting of NSCs at a defined time and location with high penetrance. It avoids potential undesired effects originating from gene knock-out in NSCs in other regions such as the brain that occur when using the Cre-LoxP system. It also avoids developmental effects originating from a persistent knock-out, allowing the study of gene function focused on regeneration. In addition,...

The spinal cord of most vertebrates is unable to regenerate following injury, leading to permanent disability. Several salamanders, such as the axolotl, are notable exceptions. The axolotl can fully regenerate a structurally identical spinal cord and completely restore spinal cord function. Much of the regenerative capability of the axolotl spinal cord is due to ependymal cells. These cells line the central canal, and unlike those in mammals, axolotl ependymal cells remain as neural stem cells (NSCs) post-embryonic development. After spinal cord injury (e.g., from a tail amputation), these NSCs proliferate to regrow the ependymal tube and differentiate to replace lost neurons1,2,3. Uncovering how axolotl spinal cord NSCs remain pluripotent and become activated after injury can provide valuable information on developing new therapeutic strategies for human patients.
Due to advances in the CRISPR-Cas9 gene knock-out technique, performing knock-outs to decipher gene function has become easier and has been shown to have broad applicability in various species, including axolotls4,5,6,7,8. The recent release of the full axolotl genome and transcriptome now allows any genomic locus to be targeted and for better assess to off-target effects9,10,11,12,13,14. Optimized protocols have been developed for knock-out and knock-in in axolotls using the CRISPR-Cas9 system15. Delivery of the CRISPR-Cas9 machinery in the form of CAS9 protein-gRNA ribonucleoprotein (RNP) has been shown to be more efficient than using Cas9 and gRNA-encoding plasmids4. This is likely due to the RNP being smaller in size than plasmid vectors, its ability to create DNA breaks immediately, and protecting of the gRNA from RNA degradation. In addition, using RNPs bypasses transcription and translation; thus, it avoids issues such as promoter strength and optimal codon usage when plasmid elements are derived from a different species.
Loss-of-function studies are one of the general approaches to investigating potential functions of genes of interest. In order to study gene function during regeneration, a knock-out should ideally be performed just before an injury to avoid effects on development. In addition, the knock-out should be restricted to both the NSCs and region of regeneration. A knock-out of the target gene in all NSCs (including those in the brain, which is the case in Cre-LoxP systems), may produce effects not related to regeneration that can confound the interpretation of results. Fortunately, the structure of the axolotl spinal cord provides a unique opportunity for time- and space-restricted knock-out in NSCs. Most of the spinal cord NSCs are in contact with the central canal and constitute the vast majority of the cells in contact with the central canal16,17. Therefore, an injection of the CAS9-gRNA complex into the central canal, followed by electroporation, allows delivery to spinal cord NSCs in a desired region at a specific time4,18,19. This protocol demonstrates how this is performed, leading to highly penetrating knock-out in the targeted spinal cord NSCs. Subsequent analysis are then performed to study the effects on regeneration and NSC behavior.

<table border="0" cellpadding="0" cellspacing="0" style="width:1355px;" width="1354">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Agarose</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">A9539</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">&#65279;Benzocaine</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">E1501-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:492px;">&#65279;Benzocaine 0.03 % (wt/vol)</td>
			<td style="width:455px;">Mix 500 ml of 10&#215; TBS, 500 ml of 400% (wt/vol) Holtfreter&#8217;s solution and 30 ml of 10% (wt/vol) benzocaine stock solution. Fill up the volume to 10 L with dH2O. &#65279;The solution can be stored at room temperature for up to 6 months.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:492px;">&#65279;Benzocaine 10 % (wt/vol)</td>
			<td style="width:455px;">Mix 50 g of benzocaine in 500 ml of 100% (vol/vol) ethanol. The solution can be stored at room temperature for up to 12 months.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Borosilicate glass capillaries &#65279;1.2 mm O.D., 0.94 mm I.D.</td>
			<td style="width:455px;">Stutter Instrument&#160;</td>
			<td style="width:241px;">BF120-94-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:492px;">CaCl2&#183;2H2O</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;102382</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:492px;">CAS9 buffer, 10x</td>
			<td style="width:455px;">Mix 200 mM HEPES and 1.5 M KCl in RNase-free water. Adjust pH to 7.5. Filter sterilize, aliquot and store at &#8722;20 &#176;C for up to 24 months</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">&#65279;CAS9-NLS protein&#160;&#160;</td>
			<td style="width:455px;">PNA Bio</td>
			<td style="width:241px;">CP03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Cell culture dishes, 10cm</td>
			<td style="width:455px;">Falcon</td>
			<td style="width:241px;">351029</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Dumont #5 - Fine Forceps</td>
			<td style="width:455px;">Fine Scientific Instruments</td>
			<td style="width:241px;">11254-20</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Electroporator</td>
			<td style="width:455px;">Nepa Gene&#160;</td>
			<td style="width:241px;">NEPA21</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:492px;"></td>
			<td style="width:455px;">BEX</td>
			<td style="width:241px;">Pulse Generator CUY21EDIT II</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Fast Green FCF</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">F7252-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:492px;">Fast Green FCF Solution, 5x</td>
			<td style="width:455px;">Dissolve 12.5 mg of Fast Green FCF powder in 10 mL of 1&#215; PBS.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Flaming/Brown Micropipette Puller&#160;</td>
			<td style="width:455px;">Stutter Instrument&#160;</td>
			<td style="width:241px;">P-97</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:492px;">&#65279;Holtfreter&#8217;s solution 400% (wt/vol)&#160;</td>
			<td style="width:455px;">&#65279;Dissolve 11.125 g of MgSO4&#183;7H2O, 5.36 g of CaCl2&#183;2H2O, 158.4 g of NaCl and 2.875 g of KCl in 10 L of dH2O. The solution can be stored at room temperature for up to 6 months.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">KCl</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;104936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:492px;">MgSO4&#183;7H2O</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;105886</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Microloader pipette tips</td>
			<td style="width:455px;">Eppendorf</td>
			<td style="width:241px;">5242956003</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Micromanipulator&#160;</td>
			<td style="width:455px;">Narishige</td>
			<td style="width:241px;">MN-153&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">NaCl</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;106404</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Pneumatic PicoPump</td>
			<td style="width:455px;">World Precision Instruments&#160;</td>
			<td style="width:241px;">SYS-PV830</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Ring Forceps</td>
			<td style="width:455px;">Fine Scientific Instruments</td>
			<td style="width:241px;">11103-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Stereomicroscope</td>
			<td style="width:455px;">Olympus</td>
			<td style="width:241px;">SZX10&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Tris base</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">&#65279;T6066</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="112">
			<td height="112" style="height:112px;width:492px;">Tris-buffered saline, 10x</td>
			<td style="width:455px;">&#65279;Dissolve 24.2 g of Tris base and 90 g of NaCl in 990 ml of dH2O. Adjust pH to 8.0 by adding 10 ml of 37% (vol/vol) HCl. The solution can be stored at room temperature for up to 6 months.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:492px;">Tweezers w/Variable Gap 2 Round Platinum Plate Electrode, 10mm diameter</td>
			<td style="width:455px;">Nepa Gene&#160;</td>
			<td style="width:241px;">&#65279;CUY650P10</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

direct gene knock-out of axolotl spinal cord neural stem cells via electroporation of cas9 protein-grna complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Injection and electroporation of CAS9-gRNA complex against Sox2 into the axolotl spinal cord central canal led to a massive loss of SOX2 immunoreactivity in a majority of spinal cord NSCs, with gRNA against Tyrosinase (Tyr) as a control (Figure 2A). B3-tubulin (stained with TUJ1)&#160;is a marker for neurons and was not expressed in NSCs, and SOX2- TUJ1- cells surrounding the central canal were considered to be cells harboring Sox2 deletions. Quantification...

Watch this Scientific Journal Video about Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes at JoVE.com

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

Gene direto knock-out de células-tronco neurais da medula espinhal Axolotl via eletroporação de complexos de proteína-gRNA CAS9

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

direct-gene-knock-out-axolotl-spinal-cord-neural-stem-cells-via

Research

JoVE Journal

Genetics

7.9K visualizações.  South China Normal University. Este protocolo permite o tempo rápido e altamente penetrante e eliminações genéticas com restrição de espaço em células-tronco neurais da medula espinhal axolotl para estudo dos mecanismos moleculares de regeneração da medula espinhal. Esta técnica permite o estudo da função genética em células-tronco neurais, sua regeneração médica sem ser confundida por efeitos de desenvolvimento ou efeitos de outros tipos celulares. Células-tronco neurais axolotil contribuem em grande pa...

Vídeo: Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

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.

Geração independente dependente da seleção e de genes independentes de CRISPR / Cas9 em células de mamíferos

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

Genética

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.

Uma metodologia padrão para examinar a mutagenicidade no local como uma função de ponto de mutação reparação catalisada por CRISPR/Cas9 e SsODN em células humanas

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

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

Microinjeção de proteína CRISPR/Cas9 em bagre do canal, Ictalurus punctatus, embriões para Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Um protocolo para a produção de Lentivirus vetores Integrase deficiente CRISPR/Cas9-mediada Gene nocaute em dividir células

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Disrupção do Gene altamente eficiente de células progenitoras hematopoiéticas murino e humano por CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

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.

Proteína endógena de marcação em humanos induzida por células-tronco pluripotentes usando 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 ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Preparação e modificação genética de primatas não humanos tronco hematopoiéticas e células progenitoras

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

Usando o Sniper-Cas9 para minimizar efeitos fora do alvo de CRISPR-Cas9 sem perda de atividade no alvo através de evolução dirigida

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

CRISPR/Cas9 Ribonucleoproteína-mediada por Gene preciso de edição por tubo Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

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.

Geração de modificações genómicas definidas usando CRISPR-CAS9 em células-tronco pluripotentes humanas

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