Application of Cas12j for Streptomyces Editing

Abstract

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.

Keywords: CRISPR-Cas; Cas12j-2; Streptomyces; genome editing; natural product discovery.

Figure 1

Representation of Cas proteins used in this study. Comparison of SpCas9 (PDB: 4OO8), FnCas12a (PDB: 6I1L), and AsCas12j-2 (PDB: 7LYS). Structures are shown to the same scale.

Figure 2

(A) Schematic homology flanks used for Streptomyces sp. A34053 editing along with (B) heat map representation of secondary metabolite production in wild-type and edited mutants for Cluster 5, 52, and 74 (Table 2). (A) Homology flanks (blue) with insertion of promoters are designed as the figures, where each flank is ~2 kb in length and 97 bps (kasO*p) or 778 bps (kasO*p with p8 promoter) is inserted at the specified sites. The sites of the protospacer are also indicated on the genome with arrows. Legend of genes on the genome annotation—grey: domain of unknown function; orange: transporter; green: regulator; red: biosynthetic enzymes. (B) Representative LCMS spectrum comparison between extracts of mutants and wild type. RT: retention time (min). MS spectra are shown in Figures S6 and S7.