Oca2targeting using CRISPR/Cas9 in the Malawi cichlidAstatotilapia calliptera

Abstract

Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterizing the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera , a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, probably a result of non-homologous end joining, and a large deletion in the 3′ untranslated region due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.

East African cichlid fishes

“…Gene function in cichlids has traditionally been studied using pharmacological manipulations using small molecules, such as agonists or antagonists of the target gene or gene pathways [ 106 , 173 , 195 , 196 ]. More recently, CRISPR/Cas9 has been used and shown to work at high efficiency in several cichlid species ( Astatotilapia burtoni , Astatotilapia calliptera , Pundamilia nyererei , Oreochromis niloticus ) to target both coding and non-coding sequences [ 29 , 69 , 197 – 199 ]. DNA, RNA, proteins, or combinations thereof are injected at the single- or two-cell stage into oocytes using microinjection setups (Fig.…”

“…There are several established inbred strains of cichlid species that have been in captivity for decades, such as certain populations of A. burtoni and N. brichardi [ 2 ] . Moreover, there are amelanistic lines that have been generated using CRISPR–Cas9 [ 29 , 199 ] or derived from spontaneous mutations [ 205 ]. Importantly, most cichlid species can be kept in the laboratory, and most are easily collected in the field with the appropriate export permits.…”

Morphological and temporal variation in early embryogenesis contributes to species divergence in Malawi cichlid fishes

“…We determined the chronology of gastrulation and somitogenesis and the total number of generated somites in each species, since somite stage (ss) is a commonly used index to stage-match embryos. Due to its established experimental amenability (Clark et al, 2022), we used Astatotilapia calliptera (AC) embryos to illustrate the common features of cichlid development as observed in live specimens (Figure 3). Following cleavage and blastula stages (Figure 3a and b, respectively), all species reach 20% epiboly around 26-28 hpf (Figure 2, st. 9, Figure 3c).…”

“…Insights into the genetic basis of cichlid traits have been possible due to their experimental tractability, including viability of hybrid crosses and amenability to genetic manipulations such as CRISPR-Cas9 (Albertson and Kocher, 2006;Clark et al, 2022;Juntti et al, 2013;Kocher, 2004;Li et al, 2021;Powder and Albertson, 2016). Despite these advances, the cellular and developmental mechanisms underlying cichlid morphological diversification remain unknown.…”

Developmental plasticity and variability in the formation of egg-spots, a pigmentation ornament in the cichlidAstatotilapia calliptera

“…Phylogenetic analysis shows that all Malawi cichlid species (~850 species) resulted from three separate cichlid radiations that stemmed from a generalist Astatotilapia -type ancestral lineage (Malinsky et al, 2018). As such, this species represents the ancestral bauplan of egg-spots for the Malawi cichlid radiation (Turner et al, 2021). More specifically, we focus on a population of A. calliptera from an isolated crater lake north of Lake Malawi referred to in the literature as Lake Masoko (variant spelling Massoko, as used by the German colonial administration) and known locally as Lake Kisiba (Clark et al, 2021;Turner et al, 2019).…”