Multi-Layer Controls of Cas9 Activity Coupled With ATP Synthase Over-Expression for Efficient Genome Editing in Streptomyces.

Kai Wang,Xu-Ming Mao,Richard Burchmore,Yong-Quan Li,Xin-Ai Chen,Karl Burgess,Chen-Fan Sun,Yi-Fan Liu,Qing-Wei Zhao

doi:10.3389/fbioe.2019.00304

Abstract

Efficient genome editing is a prerequisite of genetic engineering in synthetic biology, which has been recently achieved by the powerful CRISPR/Cas9 system. However, the toxicity of Cas9, due to its abundant intracellular expression, has impeded its extensive applications. Here we constructed a genetic cassette with triple controls of Cas9 activities at transcriptional, translational and protein levels, together with over-expression of the ATP synthase β-subunit AtpD, for the efficient genome editing in Streptomyces. By deletion of actII-ORF4 in Streptomyces coelicolor as a model, we found that constitutive expression of cas9 had about 90% editing efficiency but dramatically reduced transformation efficiency by 900-fold. However, triple controls of Cas9 under non-induction conditions to reduce its activity increased transformation efficiency over 250-fold, and had about 10% editing efficiency if combined with atpD overexpression. Overall, our strategy accounts for about 30-fold increased possibility for successful genome editing under the non-induction condition. In addition, about 80% editing efficiency was observed at the actII-ORF4 locus after simultaneous induction with thiostrepton, theophylline and blue light for Cas9 activity reconstitution. This improved straightforward efficient genome editing was also confirmed in another locus redD. Thus, we developed a new strategy for efficient genome editing, and it could be readily and widely adaptable to other Streptomyces species to improve genetic manipulation for rapid strain engineering in Streptomyces synthetic biology, due to the highly conserved genetic cassettes in this genus.

Highlights

Given the high efficiency and precise targeting, CRISPR/Cas9 is regarded as the most powerful genome editing toolkit for dissection of molecular mechanisms and control of gene expression (Xu and Qi, 2018)
Our hypothesis is that the reduced Cas9 activity should be beneficial for cell viability during transformation, and subsequently efficient genome editing would occur after Cas9 activity is induced for DSB coupled with homology directed recombination (HDR)
We developed an inducible genetic cassette to control the Cas9 activity in Streptomyces

Summary

Introduction

Given the high efficiency and precise targeting, CRISPR/Cas is regarded as the most powerful genome editing toolkit for dissection of molecular mechanisms and control of gene expression (Xu and Qi, 2018) It has exhibited great potential in synthetic biology for cell reprogramming, protein engineering and circuitry design (Fellmann et al, 2017; Ho and Chen, 2017), and for microbial metabolic engineering and manipulation of genetically intractable microorganisms (Cho et al, 2018b; Shapiro et al, 2018). Many efforts have been taken to reduce Cas toxicity by controlling its endogenous nuclease activity (Richter et al, 2017) These include transient delivery of Cas9/sgRNA ribonucleoprotein to avoid over-accumulation of intracellular Cas during cell propagation (DeWitt et al, 2017), and chemically-inducible expression of Cas to reduce off-target cleavages and facilitate precise genome editing (Cao et al, 2016). Post-translational control of Cas by fusion of Cas to ERT2 (Liu et al, 2016) or Cas splits to FKBP and FRB (Zetsche et al, 2015), respectively, can sequester Cas in the cytoplasm, and trans-localization of Cas into nuclei for genome editing is efficiently induced by 4-hydroxytamoxifen (Liu et al, 2016) or rapamycin (Zetsche et al, 2015)

Methods

Results

Conclusion