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

Yao Zu,Weiming Li,Xushuai Zhang,Jianfeng Ren,Qinghua Zhang,Zhe Zhu,Liang Jia,Xuehong Dong

doi:10.1038/srep23496

Yao Zu, Weiming Li + Show 6 more

Open Access

https://doi.org/10.1038/srep23496

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Abstract

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.

Highlights

Lampreys and hagfishes are sole surviving lineages of jawless vertebrates, or agnathans
We examined three Cas[9] proteins for their efficiency in altering lamprey alleles: 1) ZCas[9], the Streptococcus pyogenes Cas[9] optimized for zebrafish codon usage with an SV40 nuclear localization signal (NLS) at the 5′ end and a nucleoplasmin NLS at the 3′ end29; 2) LCas[] and 3) LCas[], both of which were optimized according to the codon usage determined from 15,790 coding sequences of the sea lamprey draft genome (Fig. S1, Table S1)
Lamprey species are used extensively in development and evolution studies[3]. These species are not readily amenable to either forward genetics or reverse genetics, the traditional approaches that rely on the mutations recovered and maintained through germ line transmission

Summary

Introduction

Lampreys and hagfishes are sole surviving lineages of jawless vertebrates, or agnathans. We reasoned that a method that efficiently disrupts both alleles of genomic loci in the founder (F0) generation would be amenable for studies of gene function in lamprey species. A series of designer nucleases targeting specific endogenous DNA sequences has been developed[15,16] Among these systems, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/ CRISPR-associated proteins) system emerged as a method of choice for its simplicity and efficiency in introducing mutations of target genes in a variety of model organisms[17,18,19,20]. We applied the optimized CRISPR/Cas[9] system in five loci, wee[1], soxe[2], kctd[10], wnt7b and golden (gol or slc24a5) These genes were selected because they have confirmed functions in vertebrate development or give convenient and consistent readouts of phenotypes, or both[7,8,25,26]. We conclude that the CRISPR/Cas9-based technique is an efficient means to disrupt both alleles of endogenous genes in lamprey

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Conclusion