Developing a Base Editing System for Marine Roseobacter Clade Bacteria

Abstract

The Roseobacter clade bacteria are of great significance in marine ecology and biogeochemical cycles, and they are potential microbial chassis for marine synthetic biology due to their versatile metabolic capabilities. Here, we adapted a CRISPR-Cas-based system, base editing, with the combination of nuclease-deactivated Cas9 and deaminase for Roseobacter clade bacteria. Taking the model roseobacter Roseovarius nubinhibens as an example, we achieved precise and efficient genome editing at single-nucleotide resolution without generating double-strand breaks or requesting donor DNAs. Since R. nubinhibens can metabolize aromatic compounds, we interrogated the key genes in the β-ketoadipate pathway with our base editing system via the introduction of premature STOP codons. The essentiality of these genes was demonstrated, and for the first time, we determined PcaQ as a transcription activator experimentally. This is the first report of CRISPR-Cas-based genome editing in the entire clade of Roseobacter bacteria. We believe that our work provides a paradigm for interrogating marine ecology and biogeochemistry with direct genotype-and-phenotype linkages and potentially opens a new avenue for the synthetic biology of marine Roseobacter bacteria.

One-for-all gene inactivation via PAM-independent base editing in bacteria

“…Four edited strains have been selected, each with three biological independent colonies, including glnA edited by dCas9-AID, by dSpRY-AID, argH edited by dSpRY-AID, and tryB and aspC edited by dSpRY-AID (Figure 5). According to the WGS data, we found unexpected high frequency of off-target events comparing to previous reports using AID from P. marinus (Banno et al, 2018;Wang et al, 2023;Wei et al, 2023). All off-target editing were C-to-T or G-to-A transitions probably due to deamination, agreeing with previous reports (Rodrigues et al, 2021;Tong et al, 2019).…”

“…We observed that when the loci of the target moved to positions -19 and -20, the editing efficiencies significantly increased reaching 100% and 95.45 ± 9.09%, indicating the capacity of dSpRY-AID for improving editing performance (Figure 1C). To be noticed, we also found mixed signals in the resulting colonies at the first round of selection, which is a common issue for base editing (Wei et al, 2023;Xia et al, 2020), while strains with pure edits could be obtained via one more round of segregation (Figure S2).…”

“…Notably, base editing employs a nuclease-deactivated Cas (dCas) protein with lower toxicity to bacteria, and it does not demand on HR for the desired editing, eventually relieving the reliance on highly efficient DNA delivery strategies to enable abundant transformants surviving from a "dead-or-alive" selection (Collias et al, 2023;Xia et al, 2020). These advantages empower base editing a preferable tool for gene editing, and its capacities have been demonstrated in various bacteria, including Escherichia coli (Banno et al, 2018), cyanobacteria (Lee et al, 2023;Wang et al, 2023), acetogens (Xia et al, 2020) and marine bacteria (Wei et al, 2023).…”

Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects

“…Furthermore, V. natriegens developed a novel NT-CRISPR approach by expressing anti-Cas9 proteins to overcome Cas9-induced cell toxicity, generating up to 100% editing (deletion) efficiency [ 135 ]. Recently, advancement was reported in the utilization of CRISPR/dCas9 for precise and efficient single-nucleotide resolution genome editing in the aromatic compound-metabolizing bacterium Roseovarius nubinhibens [ 136 ]. This was achieved without inducing double-strand breaks or the provision of donor DNA.…”

“…This was achieved without inducing double-strand breaks or the provision of donor DNA. Leveraging the editing system, critical genes within the β-ketoadipate pathway unequivocally establish the indispensability of pobA , pcaH , and pcaG in the β-ketoadipate pathway for the degradation of aromatic compounds [ 136 ]. Moreover, this study revealed that the absence of pcaQ in the genome hinders the consumption of aromatic compounds.…”