Name: Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e (Video) | JoVE Journal
Uploaded: 2023-02-17T13:15:00.000+00:00
Duration: 7 min 31 s
Description: Discover PAM-less base editing using zSpRY-ABE8e for precise zebrafish disease modeling. This method enables efficient adenine-to-guanine conversion to study single-nucleotide variants and genetic mechanisms.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 or T)18. Ensure that the target adenine nucleotide is in the highly active editing window (third to ninth nucleotide along the protospacer).</li>
	</ol>
	</li>
	<li>Order EasyEdit sgRNA (EE gRNA) modified with 2&#8242;-O-methyl-3&#8242;-phosphorothioate (MS) at both ends (Figure 1). 
	NOTE: The EE gRNA should be dissolved in RNase-free water and stored at &#8722;80 &#176;C.</li>
	<li>Linearize the zSpRY-ABE8e plasmid18.
	<ol>
		<li>Linearize 6 &#181;g of the plasmid with the XbaI enzyme (Table of Materials) in a metal bath at 37 &#176;C for 3 h.</li>
		<li>Purify the linearized plasmid using the DNA purification kit (see Table of Materials) according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Transcribe the mRNA using the in vitro transcription kit (Table of Materials).
	<ol>
		<li>Transcribe the zSpRY-ABE8e mRNA with the linearized plasmid as template according to the manufacturer&#39;s instructions. 
		NOTE: All operations involving mRNA and gRNA require the use of RNase-free laboratory supplies.</li>
	</ol>
	</li>
	<li>Use the RNA purification kit (Table of Materials) to purify the mRNA according to the manufacturer&#39;s instructions. 
	NOTE: Before eluting, the ethanol must be dried to avoid cytotoxicity, and the mRNA should be stored at &#8722;80 &#176;C if not used immediately.</li>
</ol>
2. Preparing microinjection glass capillaries
<ol>
	<li>Set up the micropipette puller system, and preheat for at least 30 min.</li>
	<li>Run a ramp test to determine the heat value needed to melt the borosilicate glass capillaries (with 1 mm outer diameter and 0.58 mm inner diameter) according to the manufacturer&#39;s instructions.</li>
	<li>Select a program, and click on the number on the keyboard to enter the parameter values. Set the following parameters to pull the glass capillaries: heat = ramp test value, pull = 50, velocity = 100, time = 50, and pressure = 30.</li>
	<li>Install the capillary tube, ensuring it is in the groove. Press PULL START/STOP to run the program.</li>
	<li>Preserve the pulled capillaries by inserting them into foam that contains gaps, keeping the needle suspended.</li>
</ol>
3. Microinjection of the zSpRY-ABE8e mRNA and EE gRNA mixture into zebrafish embryos
<ol>
	<li>Set up breeding tanks the night before the injection. Insert a divider, place two females and one male AB strain zebrafish (3-18 months old) in each tank, and cover with the lid. Refer to previous publications for gender distinctions39. 
	NOTE: To obtain more embryos, select fish that have not been breeding for at least 1 week.</li>
	<li>The next morning before the injection, thaw the zSpRY-ABE8e mRNA and EE gRNA on ice. Mix the mRNA and gRNA to final concentrations of 400 ng/&#181;L and 200 ng/&#181;L, respectively.
	<ol>
		<li>Perform the entire procedure on ice using RNase-free tips and tubes, and handle with gloves.</li>
	</ol>
	</li>
	<li>Configure the pneumatic microinjector. Close the partial pressure valve of the air pump and open the main valve first, unscrew the gas cylinder, and adjust the pressure of the partial pressure valve to 0.2 MPa/29 psi.
	<ol>
		<li>Turn on the pneumatic microinjector, set the mode to TIMER, and adjust the initial parameter pressure value to 20.0 psi and the TIMER value to 0.040 s.</li>
	</ol>
	</li>
	<li>Use fine forceps to carefully break the needles of the pulled glass capillaries under a stereomicroscope, making sure the needles are cut at an angle to facilitate the piercing of the chorion and the egg. 
	NOTE: The break point of the needle needs to be in the right place and be strong enough to pierce the egg yet thin enough to minimize damage to the egg.</li>
	<li>Add the mixture of zSpRY-ABE8e mRNA and gRNA to the tip of a glass capillary using a microloader pipette tip, avoiding bubbles in the capillary. Attach the needle to the micromanipulator, and configure the pressure and TIMER value of the injector to ensure 2 nL of mixture is produced per injection using a microcap.</li>
	<li>Unplug the dividers of the breeding tanks, and allow the fish to breed for 10-15 min. Collect the eggs using a strainer, and examine the cell stage and quality of the eggs under a stereomicroscope. Select the eggs in the single-cell state for injection to improve the editing efficiency.</li>
	<li>Line the eggs on a 1.5% agarose injection plate, and inject 2 nL of the mixture into the cell, not the yolk, of the embryos under a microscope to improve the editing efficiency. Retain at least 15 eggs as the control group.</li>
	<li>Use 1x E3 buffer to collect the eggs in Petri dishes, and place them in an incubator at 28 &#176;C. Remove the dead eggs, and change the culture buffer every 12 h until 48 hours post fertilization (hpf).</li>
</ol>
4. Efficiency analysis of base editing with EditR
<ol>
	<li>At 48 hpf, collect three pools of six randomly selected injected embryos and six randomly selected control embryos into PCR tubes respectively. Use a pipette to aspirate the residual E3 buffer.</li>
	<li>Add 40 &#181;L of 50 mM NaOH into the PCR tubes. Put the tubes in a metal bath at 95 &#176;C for 20 min, and then vortex for 15 s.</li>
	<li>Add 4 &#181;L of 1 M Tris&#183;HCl (pH 8.0) into each tube to neutralize the NaOH. Briefly centrifuge at 2,000 x g for 10 s at room temperature to remove droplets from the tube wall. Collect the supernatant into new PCR tubes, and store them at &#8722;20 &#176;C.</li>
	<li>Design appropriate primers upstream and downstream of the gRNA target sites. Use 1 &#181;L of the above supernatant as template for the PCR reactions of 50 &#181;L volume. The primers used for the PCR in this study are listed in Supplementary Table 1.</li>
	<li>Perform a DNA agarose gel electrophoresis to ensure correct and specific bands, followed by Sanger sequencing as previously described40.</li>
	<li>Analyze the Sanger sequencing data with the EditR (1.0.10) program41.
	<ol>
		<li>Upload the sequencing file (ab1 format) into the &#34;Upload .ab1 File&#34; box.</li>
		<li>Enter the 20 bp gRNA sequence in 5&#8242;-3&#8242; orientation into the &#34;Enter gRNA sequence&#34; box.</li>
		<li>Open the sequencing file (ab1 format), and confirm the base positions where the 20 bp gRNA sequence starts and ends. Enter the corresponding base position in the &#34;5&#8242; start&#34; and &#34;3&#8242; end&#34; boxes.</li>
		<li>Click on Predicted Editing to obtain the base editing efficiency. Check the quality of the sequencing data near the gRNA target sequence to avoid disorderly peaks or indels. 
		NOTE: In general, the disorderly peaks can be effectively removed by adjusting the PCR annealing temperature.</li>
	</ol>
	</li>
</ol>

<ol><li>Eichler, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Completing+the+map+of+human+genetic+variation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Completing the map of human genetic variation.</a> Nature. 447 (7141), 161-165 (2007).</li>
<li>Koukouli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nicotine+reverses+hypofrontality+in+animal+models+of+addiction+and+schizophrenia%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Nicotine reverses hypofrontality in animal models of addiction and schizophrenia.</a> Nature Medicine. 23 (3), 347-354 (2017).</li>
<li>Kalueff, A. V., Stewart, A. M., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+an+emerging+model+for+studying+complex+brain+disorders%5BTitle%5D)+AND+%22Trends+in+Pharmacological+Sciences%22%5BJournal%5D)">Zebrafish as an emerging model for studying complex brain disorders.</a> Trends in Pharmacological Sciences. 35 (2), 63-75 (2014).</li>
<li>Anzalone, A. V., Koblan, L. W., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+editing+with+CRISPR-Cas+nucleases%2C+base+editors%2C+transposases+and+prime+editors%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.</a> Nature Biotechnology. 38 (7), 824-844 (2020).</li>
<li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas+systems+for+editing%2C+regulating+and+targeting+genomes%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nature Biotechnology. 32 (4), 347-355 (2014).</li>
<li>Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmable+editing+of+a+target+base+in+genomic+DNA+without+double-stranded+DNA+cleavage%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.</a> Nature. 533 (7603), 420-424 (2016).</li>
<li>Nishida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+nucleotide+editing+using+hybrid+prokaryotic+and+vertebrate+adaptive+immune+systems%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.</a> Science. 353 (6305), (2016).</li>
<li>Gaudelli, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+evolution+of+adenine+base+editors+with+increased+activity+and+therapeutic+application%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Directed evolution of adenine base editors with increased activity and therapeutic application.</a> Nature Biotechnology. 38 (7), 892-900 (2020).</li>
<li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+RNA-guided+base+editing+in+rabbit%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Highly efficient RNA-guided base editing in rabbit.</a> Nature Communications. 9, 2717(2018).</li>
<li>Levy, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytosine+and+adenine+base+editing+of+the+brain%2C+liver%2C+retina%2C+heart+and+skeletal+muscle+of+mice+via+adeno-associated+viruses%5BTitle%5D)+AND+%22Nature+Biomedical+Engineering%22%5BJournal%5D)">Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses.</a> Nature Biomedical Engineering. 4 (1), 97-110 (2020).</li>
<li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+generation+of+mouse+models+of+human+diseases+via+ABE-+and+BE-mediated+base+editing%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing.</a> Nature Communications. 9, 2338(2018).</li>
<li>Landrum, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ClinVar%3A+Public+archive+of+interpretations+of+clinically+relevant+variants%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">ClinVar: Public archive of interpretations of clinically relevant variants.</a> Nucleic Acids Research. 44, 862-868 (2016).</li>
<li>Qin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precise+A%E2%80%A2T+to+G%E2%80%A2C+base+editing+in+the+zebrafish+genome%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Precise A•T to G•C base editing in the zebrafish genome.</a> BMC Biology. 16 (1), 139(2018).</li>
<li>Tanaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+targeted+single-nucleotide+editing+in+zebrafish%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">In vivo targeted single-nucleotide editing in zebrafish.</a> Scientific Reports. 8, 11423(2018).</li>
<li>Carrington, B., Weinstein, R. N., Sood, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BE4max+and+AncBE4max+are+efficient+in+germline+conversion+of+C%3AG+to+T%3AA+base+pairs+in+zebrafish%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">BE4max and AncBE4max are efficient in germline conversion of C:G to T:A base pairs in zebrafish.</a> Cells. 9 (7), 1690(2020).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmable+base+editing+of+zebrafish+genome+using+a+modified+CRISPR-Cas9+system%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.</a> Nature Communications. 8, 118(2017).</li>
<li>Rosello, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precise+base+editing+for+the+in+vivo+study+of+developmental+signaling+and+human+pathologies+in+zebrafish%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish.</a> eLife. 10, 65552(2021).</li>
<li>Liang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SpG+and+SpRY+variants+expand+the+CRISPR+toolbox+for+genome+editing+in+zebrafish%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish.</a> Nature Communications. 13, 3421(2022).</li>
<li>Qin, W., Lu, X., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmable+base+editing+in+zebrafish+using+a+modified+CRISPR-Cas9+system%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Programmable base editing in zebrafish using a modified CRISPR-Cas9 system.</a> Methods. 150, 19-23 (2018).</li>
<li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+CRISPR%2FCas9+genome+editing+capacity+in+zebrafish+using+SaCas9%5BTitle%5D)+AND+%22G3%22%5BJournal%5D)">Expanding CRISPR/Cas9 genome editing capacity in zebrafish using SaCas9.</a> G3. 6 (8), 2517-2521 (2016).</li>
<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+editing+in+zebrafish+by+ScCas9+recognizing+NNG+PAM%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Genome editing in zebrafish by ScCas9 recognizing NNG PAM.</a> Cells. 10 (8), 2099(2021).</li>
<li>Kleinstiver, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+CRISPR-Cas9+nucleases+with+altered+PAM+specificities%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Engineered CRISPR-Cas9 nucleases with altered PAM specificities.</a> Nature. 523 (7561), 481-485 (2015).</li>
<li>Liu, K., Petree, C., Requena, T., Varshney, P., Varshney, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+the+CRISPR+toolbox+in+zebrafish+for+studying+development+and+disease%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">Expanding the CRISPR toolbox in zebrafish for studying development and disease.</a> Frontiers in Cell and Developmental Biology. 7, 13(2019).</li>
<li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li>
<li>Hu, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolved+Cas9+variants+with+broad+PAM+compatibility+and+high+DNA+specificity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Engineered+CRISPR-Cas9+nuclease+with+expanded+targeting+space%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Genome+editing+in+plants+by+engineered+CRISPR-Cas9+recognizing+NG+PAM%5BTitle%5D)+AND+%22Nature+Plants%22%5BJournal%5D)">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/pubmed?term=((Plant+genome+editing+using+xCas9+with+expanded+PAM+compatibility%5BTitle%5D)+AND+%22Journal+of+Genetics+and+Genomics%22%5BJournal%5D)">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/pubmed?term=((PAM-less+plant+genome+editing+using+a+CRISPR-SpRY+toolbox%5BTitle%5D)+AND+%22Nature+Plants%22%5BJournal%5D)">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/pubmed?term=((SpRY+greatly+expands+the+genome+editing+scope+in+rice+with+highly+flexible+PAM+recognition%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">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. T., Christie, K. A., Whittaker, M. N., Kleinstiver, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unconstrained+genome+targeting+with+near-PAMless+engineered+CRISPR-Cas9+variants%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.</a> Science. 368 (6488), 290-296 (2020).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+editing+mediated+by+SpCas9+variants+with+broad+non-canonical+PAM+compatibility+in+plants%5BTitle%5D)+AND+%22Molecular+Plant%22%5BJournal%5D)">Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.</a> Molecular Plant. 14 (2), 352-360 (2021).</li>
<li>Vicencio, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+editing+in+animals+with+minimal+PAM+CRISPR-Cas9+enzymes%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes.</a> Nature Communications. 13, 2601(2022).</li>
<li>Rosello, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disease+modeling+by+efficient+genome+editing+using+a+near+PAM-less+base+editor+in+vivo%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Disease modeling by efficient genome editing using a near PAM-less base editor in vivo.</a> Nature Communications. 13, 3435(2022).</li>
<li>Cornean, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precise+in+vivo+functional+analysis+of+DNA+variants+with+base+editing+using+ACEofBASEs+target+prediction%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.</a> eLife. 11, 72124(2022).</li>
<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+silencing+by+adenine+base+editor-mediated+start+codon+mutation%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">Efficient gene silencing by adenine base editor-mediated start codon mutation.</a> Molecular Therapy. 28 (2), 431-440 (2020).</li>
<li>Hanna, R. E., Doench, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+analysis+of+CRISPR-Cas+experiments%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Design and analysis of CRISPR-Cas experiments.</a> Nature Biotechnology. 38 (7), 813-823 (2020).</li>
<li>Doench, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+sgRNA+design+to+maximize+activity+and+minimize+off-target+effects+of+CRISPR-Cas9%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.</a> Nature Biotechnology. 34 (2), 184-191 (2016).</li>
<li>Kossack, M. E., Draper, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+regulation+of+sex+determination+and+maintenance+in+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Current+Topics+in+Developmental+Biology%22%5BJournal%5D)">Genetic regulation of sex determination and maintenance in zebrafish (Danio rerio).</a> Current Topics in Developmental Biology. 134, 119-149 (2019).</li>
<li>Crossley, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+for+Sanger+sequencing+and+molecular+assay+monitoring%5BTitle%5D)+AND+%22Journal+of+Veterinary+Diagnostic+Investigation%22%5BJournal%5D)">Guidelines for Sanger sequencing and molecular assay monitoring.</a> Journal of Veterinary Diagnostic Investigation. 32 (6), 767-775 (2020).</li>
<li>Kluesner, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EditR%3A+A+method+to+quantify+base+editing+from+Sanger+sequencing%5BTitle%5D)+AND+%22The+CRISPR+Journal%22%5BJournal%5D)">EditR: A method to quantify base editing from Sanger sequencing.</a> The CRISPR Journal. 1 (3), 239-250 (2018).</li>
<li>Gripp, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diamond-Blackfan+anemia+with+mandibulofacial+dystostosis+is+heterogeneous%2C+including+the+novel+DBA+genes+TSR2+and+RPS28%5BTitle%5D)+AND+%22American+Journal+of+Medical+Genetics+Part+A%22%5BJournal%5D)">Diamond-Blackfan anemia with mandibulofacial dystostosis is heterogeneous, including the novel DBA genes TSR2 and RPS28.</a> American Journal of Medical Genetics Part A. 164 (9), 2240-2249 (2014).</li>
<li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li>
<li>Ramakrishna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+disruption+by+cell-penetrating+peptide-mediated+delivery+of+Cas9+protein+and+guide+RNA%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.</a> Genome Research. 24 (6), 1020-1027 (2014).</li>
<li>Fu, Y., Reyon, D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+genome+editing+in+human+cells+using+CRISPR%2FCas+nucleases+and+truncated+guide+RNAs%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.</a> Methods in Enzymology. 546, 21-45 (2014).</li>
</ol>

The authors declare no conflicts of interest.

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><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&amp;nbsp;</td><td>Harvard Apparatus</td><td>BS4 30-0016</td><td></td></tr><tr><td>Cell culture dishes&amp;nbsp;</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&amp;nbsp;</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 &amp;mu;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&amp;nbsp;</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&amp;nbsp;</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>mMACHINE kit&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;middot;HCl used for genome extraction</td></tr><tr><td>&lt;em&gt;XbaI&lt;/em&gt;</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

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...

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 ...

efficient-pam-less-base-editing-for-zebrafish-modeling-human-genetic

Research

JoVE Journal

Biology

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.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microbead Implantation in the Zebrafish Embryo

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Manipulation of Gene Function in Mexican Cavefish

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

Discover PAM-less base editing using zSpRY-ABE8e for precise zebrafish disease modeling. This method enables efficient adenine-to-guanine conversion to study single-nucleotide variants and genetic mechanisms.

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

Explore More Videos