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Systematic Genome Editing of the Genes on Zebrafish Chromosome by...
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Systematic Genome Editing of the Genes on Zebrafish Chromosome by CRISPR/Cas9

A special issue of Biology (ISSN 2079-7737). This special issue belongs to the section "Genetics and Genomics".

Deadline for manuscript submissions: closed (31 May 2022) | Viewed by 13472

Special Issue Editors

Dr. De-Li Shi

E-Mail Website
Guest Editor
Laboratory of Developmental Biology, Institut de Biologie Paris-Seine, Sorbonne University, 9 quai Saint Bernard, 75252 Paris CEDEX, France
Interests: genomic features; developmental biology; zebrafish; early embryo; genome editing; CRISPR/Cas9; Wnt signaling; gastrulation; myogenesis; morphogenesis
Special Issues, Collections and Topics in MDPI journals
Dr. Bo Zhang

E-Mail Website
Guest Editor
Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, Peking University Genome Editing Research Center, College of Life Sciences, Peking University, Beijing 100871, China
Interests: zebrafish; genome editing; CRISPR/Cas system; organogenesis; heart regeneration; functional genomics; single-cell transcriptomics; disease modeling

Special Issue Information

Dear Colleagues,

The CRISPR/Cas9 system is a powerful tool in genome editing, with a wide range of potential applications. It has been used successfully in a variety of species, from invertebrates to mammals, making gene editing faster and more accessible than ever. This technology has produced an enormous effect on genetic research. Zebrafish has become particularly attractive for understanding regulatory gene functions and for modeling human diseases. The number of mutant and transgenic lines created by CRISPR/Cas9 is rapidly increasing in this commonly used animal model, which should help to illustrate the genotype–phenotype relationship in the future. It is expected that further improvements of CRISPR/Cas9-mediated conditional knockouts and transgenesis should strongly impact the systematic screening of gene functions and imaging-based cell lineage tracing during embryonic and post-embryonic development.

This Special Issue welcomes submissions of original research and review manuscripts focusing on the application of CRISPR/Cas9 technology and related molecular tools to genome editing in zebrafish, including but not limited to gene targeting and technical development or improvement. The aim is to provide an overview of the exciting advance and the strong potential for systematic analysis of gene functions in development and disease.

Dr. De-Li Shi
Dr. Bo Zhang

Guest Editors

Manuscript Submission Information

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Genetics
  • Knock-in
  • Zebrafish
  • Knock-out
  • Phenomics
  • Transgenesis
  • Chromosome
  • Disease model
  • Gene function
  • Regeneration
  • CRISPR/Cas9
  • Genome editing
  • Developmental biology
  • Lineage tracing

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Published Papers (3 papers)

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17 pages, 2367 KiB  
Article
ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish
by Bingzhou Han, Yage Zhang, Yang Zhou, Biao Zhang, Christopher J. Krueger, Xuetong Bi, Zuoyan Zhu, Xiangjun Tong and Bo Zhang
Biology 2022, 11(3), 411; https://doi.org/10.3390/biology11030411 - 8 Mar 2022
Cited by 13 | Viewed by 4390
Abstract
In zebrafish, RNA-guided endonucleases such as Cas9 have enabled straightforward gene knockout and the construction of reporter lines or conditional alleles via targeted knockin strategies. However, the performance of another commonly used CRISPR system, Cas12a, is significantly limited due to both the requirement [...] Read more.
In zebrafish, RNA-guided endonucleases such as Cas9 have enabled straightforward gene knockout and the construction of reporter lines or conditional alleles via targeted knockin strategies. However, the performance of another commonly used CRISPR system, Cas12a, is significantly limited due to both the requirement of delivery as purified protein and the necessity of heatshock of injected embryos. To explore the potential of CRISPR/Cas12a-mediated genome editing and simplify its application in zebrafish, we took advantage of the recently reported mRNA-active ErCas12a and investigated its efficacy for the knockin of large DNA fragments, such as fluorescent reporter genes. For knockin via either microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ) pathways, ErCas12a-injected embryos with a brief heatshock displayed comparable knockin efficiency with Cas9 injection. Through the fusion of T5 exonuclease (T5exo) to the N-terminus of ErCas12a (T5exo-ErCas12a), we further demonstrated high efficiency gene knockout and knockin at a normal incubation temperature, eliminating the embryo-damaging heatshock step. In summary, our results demonstrate the feasibility of ErCas12a- and T5exo-ErCas12a-mediated genome manipulation under simplified conditions, and further expand the genome editing toolbox for various applications in zebrafish. Full article
(This article belongs to the Special Issue Systematic Genome Editing of the Genes on Zebrafish Chromosome by CRISPR/Cas9)
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11 pages, 3086 KiB  
Communication
A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish
by Chong Zhang, Jiaguang Li, Imran Tarique, Yizhuang Zhang, Tong Lu, Jiasheng Wang, Aijun Chen, Fenfen Wen, Zhuoyu Zhang, Yanjun Zhang and Ming Shao
Biology 2021, 10(8), 777; https://doi.org/10.3390/biology10080777 - 16 Aug 2021
Cited by 4 | Viewed by 3227
Abstract
Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and [...] Read more.
Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(zpc:zcas9) with oocyte-specific cas9 expression, we have successfully obtained maternal or maternal–zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target dvl2 and dvl3a simultaneously and introduced this construct to the genome of Tg(zpc:zcas9) by meganuclease I-Sce I. As expected, we confirmed the existence of Mdvl2;Mdvl3a embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZdvl2;MZdvl3a embryos were also obtained by crossing the mutant carrying mosaic F0 female with dvl2+/−;dvl3a−/− male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs. Full article
(This article belongs to the Special Issue Systematic Genome Editing of the Genes on Zebrafish Chromosome by CRISPR/Cas9)
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Other

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8 pages, 1235 KiB  
Commentary
Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish
by De-Li Shi
Biology 2022, 11(1), 102; https://doi.org/10.3390/biology11010102 - 10 Jan 2022
Cited by 2 | Viewed by 4783
Abstract
Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for [...] Read more.
Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes. Full article
(This article belongs to the Special Issue Systematic Genome Editing of the Genes on Zebrafish Chromosome by CRISPR/Cas9)
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