title: Biallelic editing of a lamprey genome using the CRISPR/Cas9 system.
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.
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zu
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Xushuai
authors.initials: X
authors.lastname: Zhang
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Ren
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Xuehong
authors.initials: X
authors.lastname: Dong
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Jia
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.|Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Weiming
authors.initials: W
authors.lastname: Li
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D054643:INDEL Mutation; D007798:Lampreys / Q000235:genetics*; D059645:Mutation Rate; D010641:Phenotype