Name: efficient pam-less base editing for zebrafish modeling of human genetic disease with zspry-abe8e
Uploaded: 2023-02-17T13:15:00
Description: Watch this Scientific Journal Video about Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e at JoVE.com

We thank Barbara Garbers, PhD, from Liwen Bianji (Edanz) for editing the English text of a draft of this manuscript. This work was supported by the Key-Area Research and Development Program of Guangdong Province (2019B030335001), the National Key R&#38;D Program of China (2019YFE0106700), the National Natural Science Foundation of China (32070819, 31970782), and the Research Fund Program of Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals (PBEA2020YB05).

This study was conducted in strict compliance with the guidelines of the Care and Use Committee of the South China Normal University.
1. Preparing synthetically modified gRNA and zSpRY-ABE8e mRNA
<ol>
	<li>Design the gRNA according to previous publications37,38.
	<ol>
		<li>Preferentially select NRN as the PAM sequence, as zSpRY-ABE8e shows a higher preference for NRN PAM than NYN PAM (where R is A or G, and Y is C o...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolved+Cas9+variants+with+broad+PAM+compatibility+and+high+DNA+specificity.">Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.</a> Nature. 556 (7699), 57-63 (2018).</li><li>Nishimasu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+CRISPR-Cas9+nuclease+with+expanded+targeting+space.">Engineered CRISPR-Cas9 nuclease with expanded targeting space.</a> Science. 361 (6408), 1259-1262 (2018).</li><li>Endo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+plants+by+engineered+CRISPR-Cas9+recognizing+NG+PAM.">Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM.</a> Nature Plants. 5, 14-17 (2019).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+genome+editing+using+xCas9+with+expanded+PAM+compatibility.">Plant genome editing using xCas9 with expanded PAM compatibility.</a> Journal of Genetics and Genomics. 46 (5), 277-280 (2019).</li><li>Ren, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAM-less+plant+genome+editing+using+a+CRISPR-SpRY+toolbox.">PAM-less plant genome editing using a CRISPR-SpRY toolbox.</a> Nature Plants. 7, 25-33 (2021).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpRY+greatly+expands+the+genome+editing+scope+in+rice+with+highly+flexible+PAM+recognition.">SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition.</a> Genome Biology. 22 (1), 6 (2021).</li><li>Walton, R. 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This protocol describes the construction of a zebrafish disease model using the base editor zSpRY-ABE8e. Compared with the traditional HDR pathway for base substitution, this protocol can achieve more efficient base editing and reduce the occurrence of indels. At the same time, this protocol involves implementing the recently proposed i-Silence gene-knockout strategy in zebrafish. Taken together, zSpRY-ABE8e will enhance the application of zebrafish models in disease research.
Off-target effec...

Single-nucleotide variants (SNVs) that cause missense or nonsense mutations are the most common source of mutations in the human genome1. To determine whether a particular SNV is pathogenic, and to shed light on its pathogenesis, precise animal models are required2. Zebrafish are good human disease models, exhibiting a high degree of physiological and genetic homology with humans, a short developmental cycle, and strong reproductive ability, which is advantageous for research into pathogenic characteristics and mechanisms, as well as drug screening3.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been widely applied in the genome editing of various species, including zebrafish4. With gRNA guidance, the CRISPR/Cas9 system can generate DNA double-stranded breaks (DSBs) at the target site, which then allows single-base substitution through recombination of the target site with donor DNA templates via the homology-directed repair (HDR) pathway. However, the efficiency of this base replacement method is quite low as the cellular DNA repair process is mainly carried out by the non-homologous end-joining (NHEJ) pathway, which is usually accompanied by insertion and deletion (indel) mutations5. Fortunately, CRISPR/Cas9-based single-base editing technology significantly alleviates this problem by using base editors, which enable more efficient single-base editing without inducing DSBs. Two major classes of base editors, adenine base editors (ABEs) and cytosine base editors (CBEs), have been developed to implement base substitution editing for A&#183;T to G&#183;C and C&#183;G to T&#183;A, respectively6,7,8,9,10,11. These four types of base substitutions cover 30% of human pathogenic variants12. Both classes of base editors, including PmCDA1, BE system, CBE4max, ABE7.10, and ABE8e, have been reported to work in zebrafish, with BE4max and ABE8e especially reported to achieve high editing activity13,14,15,16,17,18,19.
Cas9 proteins from different species, including Staphylococcus aureus, Streptococcus pyogenes, and S. canis, have been implemented in zebrafish gene editing, with the Streptococcus pyogenes Cas9 (SpCas9) being used most widely20,21,22,23. However, SpCas9 can only recognize target sites with an NGG protospacer adjacent motif (PAM), which limits its editable range and can result in no suitable sequence being found near the pathogenic site of interest24. To expand the target range, a variety of SpCas9 variants have been engineered to recognize different PAMs through directed evolution and structure-guided design. However, few variants are effective in animals, especially in zebrafish, which limits the application of zebrafish in SNV-related disease research25,26,27,28. Recently, two variants of SpCas9, SpG and SpRY, with less stringent PAM restrictions (NGN for SpG and NNN for SpRY with a higher preference for NRN than NYN) have been reported to exhibit high editing activity in human cells and plants29,30,31,32. Subsequently, SpG and SpRY, as well as a number of their mediated base editors, such as SpRY-mediated CBEs and SpRY-mediated ABEs, have also been reported to work in zebrafish, which will enhance the application of zebrafish models in the mechanistic study and drug screening of SNV-related diseases18,33,34,35. Furthermore, i-Silence was proposed as an effective and accurate gene-knockout strategy through ABE-mediated start codon conversion from ATG to GTG or ACG36. The combination of the i-Silence strategy and the SpRY-mediated base editor zSpRY-ABE8e provides a new method for disease modeling18. This protocol demonstrates how to perform gene editing using zSpRY-ABE8e in zebrafish to construct a tsr2 (M1V) model using the i-Silence strategy. The editing efficiency and phenotypes that appear in zebrafish models were assessed and analyzed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>1.5% Agarose used to make an injection plate</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries&#160;</td>
			<td>Harvard Apparatus</td>
			<td>BS4 30-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes&#160;</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>ClonExpress Ultra One Step Cloning Kit</td>
			<td>Vazyme</td>
			<td>C115</td>
			<td>Kit for Infusion clone&#160;</td>
		</tr>
		<tr>
			<td>Codon optimization service</td>
			<td>Sangon Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drummond Microcaps</td>
			<td>Drummond Microcaps</td>
			<td>P1299-1PAK</td>
			<td>Length:32 mm, capacity:0.5 &#956;L</td>
		</tr>
		<tr>
			<td>EasyEdit gRNA service</td>
			<td>GenScript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Scientific Instrument</td>
			<td>11254-20</td>
			<td>Used to break meedle</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller&#160;</td>
			<td>Stutter Instrument</td>
			<td>P-97</td>
			<td>Used to pull the glass capillaries</td>
		</tr>
		<tr>
			<td>HotStart Taq PCR StarMix</td>
			<td>Genstar</td>
			<td>A033-101</td>
			<td>Used for PCR reaction</td>
		</tr>
		<tr>
			<td>Intelligent artificial climate box</td>
			<td>TENLIN</td>
			<td>PRX-1000A</td>
			<td>Used to culture zebrafish embryos</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0512</td>
			<td>Used to fix zebrafish when photographing</td>
		</tr>
		<tr>
			<td>Microloader pipette tips&#160;</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>mMACHINE kit&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mut Express II Fast Mutagenesis Kit V2</td>
			<td>Vazyme</td>
			<td>C214-01</td>
			<td>Kit for site-directed mutagenesis</td>
		</tr>
		<tr>
			<td>Pneumatic Microinjector</td>
			<td>ZGene Biotech</td>
			<td>ZGPCP-1500 PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>pT3TS-zSpCas9</td>
			<td>Addgene</td>
			<td>46757</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy FFPE kit&#160;</td>
			<td>Qiagen</td>
			<td>73504</td>
			<td>Kit for RNA purification</td>
		</tr>
		<tr>
			<td>Sanger Sequencing service</td>
			<td>Sangon Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, granular</td>
			<td>Sangon</td>
			<td>A100173-0500</td>
			<td>NaOH used for genome extraction</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus&#160;</td>
			<td>SZX10</td>
			<td>Used for photograph of phenotype</td>
		</tr>
		<tr>
			<td>SZ Series Zoom Stereo Microscope</td>
			<td>CNOPTEC</td>
			<td>SZ650</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 mMESSAGE</td>
			<td>Ambion</td>
			<td>AM1348</td>
			<td>Kit for in vitro transcription&#160;</td>
		</tr>
		<tr>
			<td>TIANprep Mini Plasmid Kit</td>
			<td>TIANGEN</td>
			<td>DP103-03</td>
			<td>Kit for plasmid extraction kit</td>
		</tr>
		<tr>
			<td>TIANquick Mini Purification Kit</td>
			<td>TIANGEN</td>
			<td>DP203-02</td>
			<td>Kit for purification for linearized plasmid</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Used to anesthetize zebrafish</td>
		</tr>
		<tr>
			<td>Tris (hydroxymethyl) aminomethane</td>
			<td>Sangon</td>
			<td>A600194-0500</td>
			<td>Component of Tris&#183;HCl used for genome extraction</td>
		</tr>
		<tr>
			<td>XbaI</td>
			<td>New England Biolabs</td>
			<td>R0145S</td>
			<td>Restriction endonuclease used for plasmid linearization</td>
		</tr>
	</tbody>
</table>

efficient pam-less base editing for zebrafish modeling of human genetic disease with zspry-abe8e

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.

The mutation of TSR2 has been reported to cause Diamond Blackfan anemia (DBA)42. Here, a DBA zebrafish model was constructed with a tsr2 (M1V) mutation using the i-Silence strategy. The adenine of the start codon of the zebrafish tsr2 was successfully converted to guanine using zSpRY-ABE8e (Figure 3).
The EditR analysis of the Sanger sequencing results showed that there was an A/G overlap at the adenine base of th...

Watch this Scientific Journal Video about Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e at JoVE.com

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

This protocol describes how to perform efficient adenine base editing without PAM limitation to construct a precise zebrafish disease model using zSpRY-ABE8e.

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but ...

This protocol allows researchers to build a number of previously unavailable zebrafish models of disease-relevant single-nucleotide variants. PAM limitations are major obstacle to creating accurate animal models, and zSpRY-ABE8e enables efficient adenine to guanine base conversion with low intermutation and relaxed PAN restriction. This tool can be used to construct accurate zebrafish models for studying the pathogenic characteristics disease mechanisms related to single-nucleotide marines, as well as drug screening for this disease.To begin, set up the micropipette puller system and preheat for at least 30 minutes. To pull the glass capillaries, select a program, set the ramp test value as heat, 50 as pull, the velocity at 100, time as 50, and pressure as 30. Next, install the capillary tube ensuring it is in the groove.Press pull start/stop"to run the program. To preserve the pulled capillaries, insert them into foam containing gaps keeping the needle suspended. For experimentation, use zSpRY-ABE8e mRNA described in the manuscript, and easy edit sgRNA modified with 2'O-methyl phosphorothioate at both ends.For the gRNA design, preferentially select NRN as the PAM sequence, as zSpRY-ABE8e shows a higher preference for NRN PAM than NYN PAM. Enter the target adenine nucleotide is in the highly active editing window from the third to the ninth nucleotide along the protospacer. Set up breeding tanks the night before the injection.Insert a divider, placing two female and one male zebrafish in each tank. Place a lid over each tank. The next morning, using RNase-free tips, tubes, and gloves, mix the thawed mRNA and EEgRNA on ice to a final concentration of 400 and 200 nanograms per microliter, respectively.To configure the pneumatic microinjector, first, close the partial pressure valve of the air pump. Then, open the main valve, unscrew the gas cylinder, and adjust the pressure of the partial pressure valve to 0.2 Megapascals. Now, the turn on the microinjector and set the mode to timer.Adjust the parameter pressure valve to 20 pounds per square inch and the timer value to 0.040 seconds. Using fine forceps, angularly break the needles of the pulled glass capillaries under a stereo microscope. Using a microloader pipette tip, add the mRNA/gRNA mixture to the tip of a glass capillary and attach the needle to the micromanipulator.Configure the pressure and timer value to produce two nanoliters mixture per injection using a microcap. After breeding the fish for 10 to 15 minutes, collect the eggs in a strainer. Examine the cell stage and egg quality under the stereo microscope.Select single-celled eggs for injection to improve the editing efficiency. Line the eggs up on a 1.5%agarose injection plate and inject two nanoliters of the RNA mixture into the cell of the embryos using a microscope. Collect the eggs in Petri dishes with E3 Buffer and incubate them at 28 degrees Celsius.Remove the dead eggs and change the culture buffer every 12 hours for 48 hours post-fertilization. After 48 hours post-fertilization, collect three pools of six randomly selected injected embryos into a PCR tube. Once six randomly selected control embryos are collected, use a pipette to aspirate the residual E3 Buffer.Add 40 microliters of 50 millimolar sodium hydroxide to the embryos. Place the tubes in a metal bath at 95 degrees Celsius for 20 minutes, and then vortex for 15 seconds. Add four microliters of one molar Tris hydrochloride at pH eight into each tube.Centrifuge the tubes at 2000g for 10 seconds at room temperature. Transfer the supernatant into a new PCR tube. Remove one microliter of the supernatant from the PCR tube and use it for Sanger sequencing.Then store the remaining supernatant at 20 degrees Celsius. To analyze the Sanger sequencing data, begin by uploading the sequencing file into the upload ab1 File"box. Enter the 20 base pair gRNA sequence in the 5'to 3'orientation into the enter gRNA"sequence box.Enter the corresponding base position into the 5'start and 3'ends boxes. Obtain the base editing efficiency by clicking on predictive editing"and checking the quality of the sequencing data. A diamond blackfan anemia zebrafish model was constructed by converting the adenine of the start codon of Tsr2 to guanine.The EditR analysis of sequence data demonstrated an AG overlap at the adenine base of the Tsr2 start codon. The phenotype of the two day-old Tsr2 embryos showed smaller eyes compared to the control embryos. A swollen pericardium was also observed in the mutant embryos.In order to achieve efficient base editing, please strictly follow the previously mentioned guideline design principle, which is a prerequisite for the success of the experiment. Accurate models are essential for the study of single-nucleotide variant-related diseases. The development of the SprY-ABE8e provides an effective tool for accurate modeling, which helps to promote the research of disease mechanisms and treatments.

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Research

JoVE Journal

Biology

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...