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Biallelic editing of a lamprey genome using the CRISPR/Cas9 system

Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional...

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Autores principales: Zu, Yao, Zhang, Xushuai, Ren, Jianfeng, Dong, Xuehong, Zhu, Zhe, Jia, Liang, Zhang, Qinghua, Li, Weiming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804306/
https://www.ncbi.nlm.nih.gov/pubmed/27005311
http://dx.doi.org/10.1038/srep23496
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author Zu, Yao
Zhang, Xushuai
Ren, Jianfeng
Dong, Xuehong
Zhu, Zhe
Jia, Liang
Zhang, Qinghua
Li, Weiming
author_facet Zu, Yao
Zhang, Xushuai
Ren, Jianfeng
Dong, Xuehong
Zhu, Zhe
Jia, Liang
Zhang, Qinghua
Li, Weiming
author_sort Zu, Yao
collection PubMed
description Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional studies of genes in these important Evo-Devo models. Here, we report a CRISPR/Cas system optimized for lamprey genomes and use it to disrupt genomic loci in the Northeast Chinese lamprey (Lethenteron morii) with efficiencies ranging between 84~99%. The frequencies of indels observed in the target loci of golden (gol), kctd10, wee1, soxe2, and wnt7b, estimated from direct sequencing of genomic DNA samples of injected lamprey larvae, were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively. These indels often occurred in both alleles. In the CRISPR/Cas9 treatment for gol or kctd10, 38.6% or 85.3% of the targeted larvae had the respective recessive null-like phenotypes, further confirming the disruption of both loci. The kctd10 gRNA, designed against an essential functional region of Kctd10, resulted in null-like phenotypes and in-frame mutations in alleles. We suggest that the CRISPR/Cas-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches.
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spelling pubmed-48043062016-03-24 Biallelic editing of a lamprey genome using the CRISPR/Cas9 system Zu, Yao Zhang, Xushuai Ren, Jianfeng Dong, Xuehong Zhu, Zhe Jia, Liang Zhang, Qinghua Li, Weiming Sci Rep Article Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional studies of genes in these important Evo-Devo models. Here, we report a CRISPR/Cas system optimized for lamprey genomes and use it to disrupt genomic loci in the Northeast Chinese lamprey (Lethenteron morii) with efficiencies ranging between 84~99%. The frequencies of indels observed in the target loci of golden (gol), kctd10, wee1, soxe2, and wnt7b, estimated from direct sequencing of genomic DNA samples of injected lamprey larvae, were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively. These indels often occurred in both alleles. In the CRISPR/Cas9 treatment for gol or kctd10, 38.6% or 85.3% of the targeted larvae had the respective recessive null-like phenotypes, further confirming the disruption of both loci. The kctd10 gRNA, designed against an essential functional region of Kctd10, resulted in null-like phenotypes and in-frame mutations in alleles. We suggest that the CRISPR/Cas-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches. Nature Publishing Group 2016-03-23 /pmc/articles/PMC4804306/ /pubmed/27005311 http://dx.doi.org/10.1038/srep23496 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Zu, Yao
Zhang, Xushuai
Ren, Jianfeng
Dong, Xuehong
Zhu, Zhe
Jia, Liang
Zhang, Qinghua
Li, Weiming
Biallelic editing of a lamprey genome using the CRISPR/Cas9 system
title Biallelic editing of a lamprey genome using the CRISPR/Cas9 system
title_full Biallelic editing of a lamprey genome using the CRISPR/Cas9 system
title_fullStr Biallelic editing of a lamprey genome using the CRISPR/Cas9 system
title_full_unstemmed Biallelic editing of a lamprey genome using the CRISPR/Cas9 system
title_short Biallelic editing of a lamprey genome using the CRISPR/Cas9 system
title_sort biallelic editing of a lamprey genome using the crispr/cas9 system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804306/
https://www.ncbi.nlm.nih.gov/pubmed/27005311
http://dx.doi.org/10.1038/srep23496
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