CRISPR-Cas9 Gene Editing In Phaeodactylum Tricornutum And Euglena Slenderum

With the extensive research and development of CRISPR system in recent decades,CRISPR-cas9 system has become the most popular genome editing tool with its successful reports cover animals,plants,fungi,and bacteria.However,extremely low efficiency of transformation and high false positive proportion were found unexpectedly in microalgae.At present,only Chlamydomonas,P.tricornutum and Nannochloropsis had succeeded in CRISPR-cas9 applications with as low as 0.1%successful editing rate.However,in this era when biological phenomena are explained by molecular mechanisms,the development of a mature,efficient and convenient genome editing strategy is undoubtedly indispensable for the in-depth study of microalgae.This study focus on constructing the CRISPR-Cas9 gene editing platform on P.tricornutum and E.gracilis.For this purpose we used the double-vector transformation system for electrical transformation,particle bombardment and PEG-mediated protoplast transformation,tried to knock-out arginine transferase 1(Ate1)in P.tricornutum but failed to gain positive clone.Considering ate1 could be a knock-out lethal gene,we changed our target gene to a transposon named artemis whose function is unknown.After a few attempts we obtained a 5bp deletion mutant from P.tricornutum through E.coli conjugation and transformation by an all-in-one CRISPR-Cas9 vector.This lead us to reconsider the reason of failure in the past three years could be the low efficiency of sg RNA in vivo.Thus,we designed 8 sg RNA target of ate1 gene and successfully obtained a 1bp insertion mutant and a 5bp deletion mutant.In this study,seven candidate genes related to the degradation of paramylon were quantified by q RT-PCR.Based on their connection with the change curve of the paramylon content of Euglena during the 7-day culture period,two candidates,Egcel17 A and Egcel81 C,whose expression level increased significantly during the period of paramylon degradation,were selected out for afterwords knock-outexperiment to verify their functions.Also,during the whole process of the degradation of paramylon,we found that the expression levels of seven candidate genes of paramylon degradation enzymes were significantly up-regulated in batches.This observation indicates that the degradation of paramylon in Euglena is likely to be a synergistic process by a variety of functional enzymes.In conclusion,this study confirm the availability of CRISPR-Cas9 gene editing system in algae while we think the low efficiency of sg RNA in vivo could be the stumbling block rather the toxicity of Cas9 protein.Thus,using at least 8 sg RNA for each gene knock-out maybe necessary.This study will be helpful to improve the efficiency of gene editing in microalgae and provide an example for the construction of other microalgae gene editing strategy.