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 ...

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