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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 ...

South China Normal University

Mannose-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, plays an important role in innate immunity which is capable of activating the lectin pathway of the complement system and also triggering coagulation cascade system. In this study, a MASP-1 homolog (OnMASP-1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and inflammation functional levels. The open reading frame (ORF) of OnMASP-1 is 2187 bp of nucleotide sequence encoding a polypeptide of 728 amino acids. The deduced amino acid sequence has 6 characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB), one epidermal growth factor domain (EGF), two complement control protein domains (CCP) and a catalytic serine protease domain (SP). Expression analysis revealed that the OnMASP-1 was highly expressed in the liver, and widely exhibited in other tissues containing intestine, spleen and kidney. In addition, the OnMASP-1 expression was significantly up-regulated in spleen and head kidney following challenges with Streptococcus agalactiae and Aeromonas hydrophila. The up-regulations of OnMASP-1 mRNA and protein expression were also demonstrated in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnMASP-1 protein was likely to participate in the regulation of inflammatory and migration reaction by monocytes/macrophages. These results indicated that OnMASP-1, playing an important role in innate immunity, was likely to involve in host defense against bacterial infection in Nile tilapia.

Expression and functional characterization of a mannose-binding lectin-associated serine protease-1 (MASP-1) from Nile tilapia (Oreochromis niloticus) in host defense against bacterial infection.

In teleost fish, IgM B cells play important roles in innate and adaptive immunity. Different IgM B cells are detected in teleost, named IgM and IgM B cell subsets, according to the distinct expression levels of membrane IgM (mIgM). However, the study on the heterogeneity in IgM B cell subsets remains poorly understood. In this study, the comparative transcriptomic profiles of IgM, IgM and IgM from peripheral blood of Nile tilapia (Oreochromis niloticus) were carried out by using RNA-sequencing technique. A total of 6045 and 5470 differentially expressed genes (DEGs) were detected in IgM and IgM cells, respectively, as compared with IgM lymphocytes, whereas 3835 genes were differentially expressed when IgM compared to IgM cells. Analysis of the KEGG database indicated that the DEGs were enriched in immune system categories and signaling transduction and interaction in IgM vs IgM, IgM vs IgM and IgM vs IgM. Comparatively, in IgM vs IgM, GO enrichment analysis indicated that the DEGs enriched in nucleic acid binding transcription factor activity. Analysis of crucial transcription factors for B cell differentiation indicated that IgM and IgM cell clusters belonged to the different B cell subsets. The data generated in this study may provide insights into understanding the heterogeneity of IgM cells in teleost, and suggest that IgM B cells play a crucial role in innate immunity.

Comparative transcriptome analysis of the transcriptional heterogeneity in different IgM cell subsets from peripheral blood of Nile tilapia (Oreochromis niloticus).

Gene targeting by homology-directed repair (HDR) can precisely edit the genome and is a versatile tool for biomedical research. However, the efficiency of HDR-based modification is still low in many model organisms including zebrafish. Recently, long single-stranded DNA (lssDNA) molecules have been developed as efficient alternative donor templates to mediate HDR for the generation of conditional mouse alleles. Here we report a method, zLOST (zebrafish long single-stranded DNA template), which utilises HDR with a long single-stranded DNA template to produce more efficient and precise mutations in zebrafish.

CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish.

CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish.

Identification and functional characterization of CD154 in T cell-dependent immune response in Nile tilapia (Oreochromis niloticus).

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of  Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from  (ScCas9), which has a more relaxed 5'-NNG-3' PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM.

Precise genetic modifications in model organisms are essential for biomedical research. The recent development of PAM-less base editors makes it possible to assess the functional impact and pathogenicity of nucleotide mutations in animals. Here we first optimize SpG and SpRY systems in zebrafish by purifying protein combined with synthetically modified gRNA. SpG shows high editing efficiency at NGN PAM sites, whereas SpRY efficiently edit PAM-less sites in the zebrafish genome. Then, we generate the SpRY-mediated cytosine base editor SpRY-CBE4max and SpRY-mediated adenine base editor zSpRY-ABE8e. Both target relaxed PAM with up to 96% editing efficiency and high product purity. With these tools, some previously inaccessible disease-relevant genetic variants are generated in zebrafish, supporting the utility of high-resolution targeting across genome-editing applications. Our study significantly improves CRISPR-Cas targeting in the genomic landscape of zebrafish, promoting the application of this model organism in revealing gene function, physiological mechanisms, and disease pathogenesis.

Article	Year
Expression and functional characterization of a mannose-binding lectin-associated serine protease-1 (MASP-1) from Nile tilapia (Oreochromis niloticus) in host defense against bacterial infec... Fish & shellfish immunology\| PubMed ID: 31096060	2019
Comparative transcriptome analysis of the transcriptional heterogeneity in different IgM cell subsets from peripheral blood of Nile tilapia (Oreochromis niloticus). Fish & shellfish immunology\| PubMed ID: 31408730	2019
CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish. BMC genomics\| PubMed ID: 31964350	2020
Identification and functional characterization of CD154 in T cell-dependent immune response in Nile tilapia (Oreochromis niloticus). Fish & shellfish immunology\| PubMed ID: 33513438	2021
Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM. Cells\| PubMed ID: 34440868	2021
SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish. Nature communications\| PubMed ID: 35701400	2022
Author Correction: SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish. Nature communications\| PubMed ID: 36797310	2023

Fang Liang

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