Application Of CRISPR/Cas9 In Amphioxus Genome Editing

Amphioxus is a key transitional species from invertebrate to vertebrate and is treated as one of the promising models for studying the origin and evolution of vertebrate,due to its analogous but simpler organic structure,embryogenesis and genomic structure,compared to vertebrate.Although recent techniques established in amphioxus,such as consecutive breeding and induced spawning,micro-injection,gene knock-out,transgenosis and so on,have advanced researches on amphioxus,they still seem insufficient for the purpose of systematically studying amphioxus,compared to well-studied model organisms.For example,CRISPR/Cas9 is not yet accessible to amphioxus,as well as methods that generate maternal mutants and insert genetic element at target sites.In this report we firstly tried to apply CRISPR/Cas9 in amphioxus.For this,two kinds of Cas9 mRNA in vitro transcribed from pXT7-Cas9 and pCS2-nls-zCas9-nls vectors,three kinds of Cas9 protein purchased from TaKaRa,Thermo and NEB companies,was separately mixed with a sgRNA previously described as an effective target site at mCherry locus,then the mixture was injected one by one into amphioxus unfertilized eggs(or injected into wile type unfertilized eggs,then fertilized them using sperm of male mCherry transgenic individuals),finally the mutational efficiencies cost by the mixture was detected at suitable stage.The result showed that two kinds of mixture containing Cas9 mRNA failed to introduce mutation at mCherry locus,instead,three kinds of mixtures containing Cas9 protein successfully induced mutation at mCherry locus,in which Cas9 protein from TaKaRa company performed best.The further analysis indicated the reason why injecting Cas9 mRNA caused negative mutation may be the rapid degradation of sgRNA after fertilization,due to lack of protection.This was firstly proved by RT-qPCR result:The amount of sgRNA of embryos at two-cell stage just accounted for 6%of that at unfertilized egg(soon after injection).Secondly,mutation was detected when the mixture containing Cas9 mRNA and sgRNA was injected at two-cell stage.To confirm that Cas9 system is capable of inducing mutations extensively in amphioxus genome,we further test another eight sgRNAs targeting four genes.The result showed that Seven of the eight show mutational efficiencies ranging from 18.4%to 90%,and four caused specific phenotypes in the injected embryos.The results above demontrated that CRISPR/Cas9 is feasible in amphioxus genome editing.The report also tried to generate maternal mutants using Cas9.Firstly,we generated a kind of amphioxus transgenic line containing Vasa promoter specifically expressing at primordial germ cells(PGCs),mScarlet-P2A-mScarlet and Vasa 3’UTR,and similar expressional pattern was observed between mScarlet and endogenous Vasa,which proved the cloned Vasa promoter and 3’ UTR could drive specific expression of exogenous genes at amphioxus PGCs.We further obtained F0 transgenic amphioxus line containing Vasa promoter,Cas9 and Vasa 3’UTR likewise.Moreover,we also tried to directly inject Cas9/sgRNA into one of the two biggest blastomeres(each of them develops to be PGCs with 50%probability)of 4-cell stage embryos to generate maternal mutants,which,demonstrated by further study,could significantly decrease the high mortality induced by whole-mount injection and generate F0 individuals whose gamete carry mutant.For example,13 of 22 Nodal F0 chimeras showed mutational efficiency at their gamete ranging from 12.5%to 92.5%;only 1 of 32 PKD2 showed 13.2%and 2 of 3 PKD1L1 showed 38.7%and 11.3%respectively.This result demonstrated that 4-cell injection could effectively generate individuals whose PGCs carry targeted mutant.But the phenotypes of these maternal mutants still need further identification.There’re still no reports about gene knock-in in amphioxus.We tried to insert mCherry at mlc,vegl-like and Bra2 loci using ShCAST,and delete a base at NodalsgRNA2 target site using prime editor,but none of them showed detectable knock-in efficiency.