Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes

Abstract

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes has enabled high efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. Novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes enabling efficient homology directed repair (HDR)-mediated knock-in and deletion. In strains where spCas9 was nonfunctional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.

Development and Application of CRISPR/Cas in Microbial Biotechnology

“…In some organisms, however, SpCas9 showed low efficiency or toxic effect, and repressed cell growth significantly ( Ungerer and Pakrasi, 2016 ; Wendt et al, 2016 ; Jiang et al, 2017 ). To solve this issue, different CRISPR systems or effector variants (e.g., Cas12a) showed high editing efficiency but lower toxicity, and were applied in those organisms ( Ungerer and Pakrasi, 2016 ; Jiang et al, 2017 ; Yeo et al, 2019 ).…”

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin

“…Protospacers were first inserted via Bbs I-mediated Golden Gate Assembly before introduction of the respective homology flanks via Gibson assembly, as previously described [3]. Detailed description of aur S5 deletion strain can also be found in [4].…”

“…Precise deletions of individual target genes without affecting intergenic regions were made using a CRISPR-Cas mediated editing strategy ([3], Figure S1-7). To delete aurS5 , we had to use a different Cas protein [4]. As described elsewhere [6], in order to activate the entire BGC for production of the corresponding analogs, luxR in these edited strains was also placed under a strong constitutive kasO * promoter [31].…”

“…Advances in genome editing and synthetic biology tools, together with natural product biosynthesis knowledge accumulated over the decades, allows us to better predict, design and build pathways towards the synthesis of natural products [2]. Earlier, we established a rapid and efficient CRISPR-Cas9 strategy for biosynthetic gene cluster (BGC) editing and activation in streptomycetes [3, 4], which opens up opportunities for combinatorial biosynthesis in native streptomycete hosts [5]. Compared to chemical syntheses, combinatorial engineering of native biosynthetic pathways allows us to design and biosynthesize structurally complex chemical analogs without traversing difficult multi-step and possibly low-yielding chemical reactions, thus facilitating elucidation of structure-activity relationships towards an optimized drug lead.…”

Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems

“…KY 40-1 ( Salem et al, 2017 ), Streptomyces sp. NRRL S-244 ( Yeo et al, 2019 ) and Streptomyces hygroscopicus SIPI-KF ( Li et al, 2018 ), because of its toxicity to the hosts.…”