CRISPR/Cas12a-Mediated Gene Editing in Geodia barretti Sponge Cell Culture

Dirk E Martens,Ana-Maria Alexandru,Rene H Wijffels,Jip Van Der Linden,Shirley A Pomponi,John L Flores Alvarez,Kylie Hesp

doi:10.3389/fmars.2020.599825

Abstract

Sponges and their associated microorganisms are the most prolific source of marine natural products, and many attempts have been made at creating a marine sponge cell line to produce these products efficiently. However, limited knowledge on the nutrients sponge cells require to grow and poor genetic accessibility have hampered progress toward this goal. Recently, a new sponge-specific nutrient medium M1 has been shown to stimulate sponge cells in vitro to divide rapidly. In this study, we demonstrate for the first time that sponge cells growing in M1 can be genetically modified using a CRISPR/Cas12a gene editing system. A short sequence of scrambled DNA was inserted using a single-stranded oligodeoxynucleotide donor template to disrupt the 2′,5′-oligoadenylate synthetase gene of cells from the boreal deep-sea sponge Geodia barretti. A blue fluorescent marker gene appeared to be inserted in an intron of the same gene and expressed by a small number of G. barretti cells. Our results represent an important step toward developing an optimized continuous sponge cell line to produce bioactive compounds.

Highlights

Developing a marine sponge cell line to produce sponge-derived chemicals in vitro has been the holy grail of sponge biotechnology ever since it was discovered that marine sponges host a wide variety of bioactive secondary metabolites in marine sponges (Pomponi, 1999; Rinkevich, 2005; Mayer et al, 2010; Schippers et al, 2011; Newman and Cragg, 2016)
Synthetic guide RNA (gRNA) were designed for both target sites in the G. barretti OAS1Ab gene, 3 gRNAs targeted the 3rd exon and 2 gRNAs targeted the 2nd intron, as only 2 suitable target sequences for Cas12a were present in the intron that would leave space for >0.5 kb US and DS homology arms (HA) (Figure 3A)
An in vitro assay in which ribonucleoprotein complexes (RNPs) of Cas12a with each of the 5 gRNAs were added to a polymerase chain reaction (PCR) product of the wild-type OAS1Ab gene was used to test how efficiently each gRNA could induce a double-stranded break (DSB) (Figure 2B, FW + RV primers)

Summary

Introduction

Developing a marine sponge cell line to produce sponge-derived chemicals in vitro has been the holy grail of sponge biotechnology ever since it was discovered that marine sponges host a wide variety of bioactive secondary metabolites in marine sponges (Pomponi, 1999; Rinkevich, 2005; Mayer et al, 2010; Schippers et al, 2011; Newman and Cragg, 2016) Many of these compounds have the potential to be developed into new drugs to combat cancer (Nuijen et al, 2000; Schwartsmann et al, 2003; Jimenez et al, 2018; Khalifa et al, 2019), inflammatory disease (Alcaraz and Paya, 2006) and infections in humans (Laport et al, 2009; Abdelmohsen et al, 2017; Liu et al, 2019a).

Methods

Results

Conclusion