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URL: https://dx.doi.org/DOI:10.17504/protocols.io.4abgsan

Authors: Mark Moosburner, Andrew Allen

Group: A.E. Allen Lab

Summary: The CRISPR-Cas9 gene mutagenesis system was adapted for the marine diatom Phaeodactylum tricornutum (CCAP-1055/1) (Figure 1). Here, Cas9 and sgRNA(s) were delivered to Phaeodactylum by bacterial-conjugation transformation on an episome, or artificial chromosome, that is stably maintained and replicated independently from and with the Phaeodactylum chromosomal DNA. Two sgRNA expression cassettes, sgRNA (1/1) and sgRNA (1/2) were cloned into the episome. The sgRNAs each contain a unique 20-nucleotide spacer sequence that , after sgRNA cassette transcription, guides the Cas9 to a nucleic acid target by complementary binding followed by Cas9 nuclease activity. Two Phaeodactylum genes, Pt_GSII (Gene ID: 51092) and Pt_cGOGAT (Gene ID: 24739), were targeted by each sgRNA, respectively. The following protocol was followed to synthesize spacer sequences and sgRNA expression cassettes

Associated Publications: Moosburner MA, Gholami P, McCarthy JK, Tan M, Bielinski VA, Allen AE, Multiplexed Knockouts in the Model Diatom by Episomal Delivery of a Selectable Cas9. Frontiers in Microbiology doi: 10.3389/fmicb.2020.00005

Affiliations: University of California, San Diego, University of California, San Diego

External URL: https://doi.org/10.3389/fmicb.2020.00005

Version: 1

Publication Date: 2019

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