Abstract
The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.
Highlights
Introduction ofCRISPR/Cas[9] plasmids into P. tricornutum.Transformation by biolistic bombardment
We present here a direct comparison between the efficiency of Cas9-mediated mutagenesis using bacterial conjugation compared to using biolistic transformation methods for the delivery of CRISPR-Cas[9] plasmids to diatom cells
The origin of transfer in the pPtPuc3m backbone allows the delivery of the plasmid to P. tricornutum cells by bacterial conjugation
Summary
Introduction ofCRISPR/Cas[9] plasmids into P. tricornutum.Transformation by biolistic bombardment. The pPtPuc3m diaCas9_sgRNA plasmid was delivered to P. tricornutum cells via conjugation from Escherichia coli DH10β cells as described by Karas and coworkers[13]. E. coli pTA-Mob competent cells (100 μl) were transformed with 200 ng of the pPtPuc3m diaCas9_sgRNA plasmid and used for conjugative delivery of the CRISPR-Cas[9] plasmid to the diatoms cells. E. coli cells (DH10β) containing pTA-MOB and CRISPR-Cas[9] plasmid (pPtPuc3m diaCas9_sgRNA) were grown in 50 ml LB media containing 50 μg/ml kanamycin and 10 μg/ml gentamicin (37 °C, 150 rpm shaking) until optical density (OD600) reaches 0.8–1.0. Once the plasmid is transformed into P. tricornutum it will replicate as an episome and remain stable if propagated in zeocin containing growth media (Supplementary Fig. 5)
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