Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

Su, Liuru; Shi, Chenggang; Huang, Xin; Wang, Yiquan; Li, Guang

doi:10.3390/genes11111311

Cited by 15 publications

(12 citation statements)

References 20 publications

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“…Significant efforts have been made to develop protocols for maintaining and spawning adult lancelets in captivity and for manipulating lancelet embryos and larvae. Thanks to these efforts, lancelets have become attractive laboratory models ( Li et al, 2013 , 2014 ; Carvalho et al, 2017b ; Shi et al, 2018 ; Su et al, 2020 ). However, one of the remaining obstacles was the absence of a widely applicable staging system guaranteeing the comparability of results obtained in different lancelet species.…”

Section: Discussionmentioning

confidence: 99%

An Updated Staging System for Cephalochordate Development: One Table Suits Them All

Carvalho

Lahaye

Yong

et al. 2021

Front. Cell Dev. Biol.

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Chordates are divided into three subphyla: Vertebrata, Tunicata, and Cephalochordata. Phylogenetically, the Cephalochordata, more commonly known as lancelets or amphioxus, constitute the sister group of Vertebrata and Tunicata. Lancelets are small, benthic, marine filter feeders, and their roughly three dozen described species are divided into three genera: Branchiostoma, Epigonichthys, and Asymmetron. Due to their phylogenetic position and their stereotypical chordate morphology and genome architecture, lancelets are key models for understanding the evolutionary history of chordates. Lancelets have thus been studied by generations of scientists, with the first descriptions of adult anatomy and developmental morphology dating back to the 19th century. Today, several different lancelet species are used as laboratory models, predominantly for developmental, molecular and genomic studies. Surprisingly, however, a universal staging system and an unambiguous nomenclature for developing lancelets have not yet been adopted by the scientific community. In this work, we characterized the development of the European lancelet (Branchiostoma lanceolatum) using confocal microscopy and compiled a streamlined developmental staging system, from fertilization through larval life, including an unambiguous stage nomenclature. By tracing growth curves of the European lancelet reared at different temperatures, we were able to show that our staging system permitted an easy conversion of any developmental time into a specific stage name. Furthermore, comparisons of embryos and larvae from the European lancelet (B. lanceolatum), the Florida lancelet (Branchiostoma floridae), two Asian lancelets (Branchiostoma belcheri and Branchiostoma japonicum), and the Bahamas lancelet (Asymmetron lucayanum) demonstrated that our staging system could readily be applied to other lancelet species. Although the detailed staging description was carried out on developing B. lanceolatum, the comparisons with other lancelet species thus strongly suggested that both staging and nomenclature are applicable to all extant lancelets. We conclude that this description of embryonic and larval development will be of great use for the scientific community and that it should be adopted as the new standard for defining and naming developing lancelets. More generally, we anticipate that this work will facilitate future studies comparing representatives from different chordate lineages.

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Section: Discussionmentioning

confidence: 99%

An Updated Staging System for Cephalochordate Development: One Table Suits Them All

Carvalho

Lahaye

Yong

et al. 2021

Front. Cell Dev. Biol.

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“…Significant efforts have been made to develop protocols for maintaining and spawning adult lancelets in captivity and for manipulating lancelet embryos and larvae. Thanks to these efforts, lancelets have become attractive laboratory models (Carvalho et al, 2017b; Su et al, 2020). However, one of the remaining obstacles was the absence of a widely applicable staging system guaranteeing the comparability of results obtained in different lancelet species.…”

Section: Discussionmentioning

confidence: 99%

An updated staging system for cephalochordate development: one table suits them all

Carvalho

Lahaye

Yong

et al. 2020

Preprint

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Background:The chordates are divided into three subphyla: Vertebrata, Tunicata and Cephalochordata. Phylogenetically, the Cephalochordata, more commonly known as lancelets or amphioxus, constitute the sister group of Vertebrata plus Tunicata. Due to their phylogenetic position and their conserved morphology and genome architecture, lancelets are important models for understanding the evolutionary history of chordates. Lancelets are small, marine filter-feeders, and the few dozen species that have so far been described have been grouped into three genera: Branchiostoma, Epigonichthys and Asymmetron.Given their relevance for addressing questions about the evolutionary diversification of chordates, lancelets have been the subjects of study by generations of scientists, with the first descriptions of adult anatomy and developmental morphology dating back to the 19 th century. Today, several different lancelet species are used as laboratory models, predominantly for developmental, molecular and genomic studies. It is thus very surprising that there is currently no universal staging system and no unambiguous nomenclature for developing lancelets. Results:We illustrated the development of the European amphioxus (Branchiostoma lanceolatum) using confocal microscopy and compiled a streamlined developmental staging system, from fertilization through larval life, with an unambiguous stage nomenclature. By tracing growth curves of the European amphioxus reared at different temperatures, we were able to show that our staging system permits the easy conversion of any developmental time into a defined 3 stage name. Furthermore, comparisons of embryos and larvae from the European amphioxus (B. lanceolatum), the Florida amphioxus (B. floridae), the Chinese amphioxus (B. belcheri), the Japanese amphioxus (B. japonicum) and the Bahamas lancelet (Asymmetron lucayanum) demonstrated that our staging system can readily be applied to other lancelet species.Conclusions: Here, we propose an updated staging and nomenclature system for lancelets. Although the detailed staging description was carried out on developing B. lanceolatum, comparisons with other lancelet species strongly suggest that both staging and nomenclature are applicable to all extant lancelets.We thus believe that this description of embryonic and larval development can be of great use for the scientific community and hope that it will become the new standard for defining and naming developing lancelets. KeywordsAmphioxus, Asymmetron lucayanum, Branchiostoma belcheri, Branchiostoma floridae, Branchiostoma japonicum, Branchiostoma lanceolatum, Confocal Microscopy, Embryonic and Larval Development, Evolution and Development, Lancelet 4

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“…Mutation efficacy was estimated by comparing band intensity between uncut and all bands [uncut/(uncut + cut)]. Band intensity was quantified using software implemented in the Tanon Gis system (Tanon, Shanghai, China) ( Su et al, 2020 ). For mutation type detection, the PCR products obtained above were further cloned into the pGEM-T easy vector (Promega, United States) (except Tesd gene, whose PCR products were ligated after digestion with Eae I).…”

Section: Methodsmentioning

confidence: 99%

“…Very recently, the CRIPSR/Cas9 system has also been applied for genome editing in B . floridae ( Su et al, 2020 ). These advances have greatly accelerated the rate of establishing amphioxus as a model organism, and enables researchers to robustly dissect the function of amphioxus genes ( Hu et al, 2017 ; Li et al, 2017 ; Ren et al, 2020 ; Zhong et al, 2020 ; Zhu et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, methods for year-round spawning induction has been developed for all three species (Benito-Gutierrez et al, 2013;Li et al, 2014bLi et al, , 2017, and TALEN-based genome editing and Tol2 transposase-mediated transgenic methods have been introduced in B. floridae and B. belcheri (Li et al, 2014a(Li et al, , 2017Shi et al, 2018). Very recently, the CRIPSR/Cas9 system has also been applied for genome editing in B. floridae (Su et al, 2020). These advances have greatly accelerated the rate of establishing amphioxus as a model organism, and enables researchers to robustly dissect the function of amphioxus genes (Hu et al, 2017;Li et al, 2017;Ren et al, 2020;Zhong et al, 2020;Zhu et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene

Zou

Shi

et al. 2021

Front. Cell Dev. Biol.

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Amphioxus is a promising model organism for understanding the origin and evolution of vertebrates due to its basal phylogenetic position among chordates. We here compared the mutation efficacy and mutation type of tail tips and gametes of amphioxus founders injected with Cas9 protein and six different sgRNAs targeting five distinct genes, and revealed a strong correlation for mutation efficacy and a mild correlation for mutation type among the two tissues. In addition, we also observed a positive relationship between gene insertions observed in tail tips and gametes of amphioxus founders injected with Tol2 transposase and two different transgenic constructs. Finally, we showed that amphioxus larvae which had their tail tips cut at the 3–4 gill-slit stage were able to recover within 6 days and developed a normal number of gonads at the adult stage, and that F0 larvae carry similar mutation efficacy and type in the posterior end to that in the tail tips after their metamorphosis. Together, these findings suggest a great potential for obtaining valid amphioxus founders with desired mutations and transgenes at as early as the early larval stage, which will certainly speed up the generation of amphioxus mutants and transgenes and make it more cost- and labor-effective.

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Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

Cited by 15 publications

References 20 publications

An Updated Staging System for Cephalochordate Development: One Table Suits Them All

An Updated Staging System for Cephalochordate Development: One Table Suits Them All

An updated staging system for cephalochordate development: one table suits them all

A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene

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