Genome-Wide Abolishment of Mobile Genetic Elements Using Genome Shuffling and CRISPR/Cas-Assisted MAGE Allows the Efficient Stabilization of a Bacterial Chassis

Abstract

The ideal bacterial chassis provides a simplified, stable and predictable host environment for synthetic biological circuits. Mutability and evolution can, however, compromise stability, leading to deterioration of artificial genetic constructs. By eliminating certain sources of instability, these undesired genetic changes can be mitigated. Specifically, deletion of prophages and insertion sequences, nonessential constituents of bacterial genomes, has been shown to be beneficial in cellular and genetic stabilization. Here, we sought to establish a rapid methodology to improve the stability of microbial hosts. The novel workflow involves genome shuffling between a mobile genetic element-free strain and the target cell, and subsequent rounds of CRISPR/Cas-assisted MAGE on multiplex targets. The power and speed of the procedure was demonstrated on E. coli BL21(DE3), a host routinely used for plasmid-based heterologous protein expression. All 9 prophages and 50 insertion elements were efficiently deleted or inactivated. Together with additional targeted manipulations (e.g., inactivation of error-prone DNA-polymerases), the changes resulted in an improved bacterial host with a hybrid (harboring segments of K-12 DNA), 9%-downsized and clean genome. The combined capacity of phage-mediated generalized transduction and CRISPR/Cas-selected MAGE offers a way for rapid, large scale editing of bacterial genomes.

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

“…CRISPR and CRISPRi have been individually harnessed to engineer genome or rewire metabolic networks in different bacteria such as cyanobacteria (Huang et al, ) and E. coli (Y. Li et al, ) to promote the production of bio‐derived chemicals. However, virtually all previous studies exploiting CRISPR or CRISPRi for E. coli engineering have used SpCas9 (Ding, Weng, Du, Chen, & Kang, ; Heo et al, ; Y. Jiang et al, ; Kiani et al, ; Liang et al, ; Liu et al; Pyne, Moo‐Young, Chung, & Chou, ; Reisch & Prather, ; Ronda, Pedersen, Sommer, & Nielsen, ; Umenhoffer et al, ; Zhao et al, ) or SpdCas9 (Cress et al, , ; Kim et al, ; S. Li, Jendresen, et al, ; Lv et al, ; Rath, Amlinger, Hoekzema, Devulapally, & Lundgren, ; Wu et al, ). Furthermore, in previous studies the CRISPRi modules were maintained in plasmids and have yet to be integrated into the chromosome using the CRISPR system, which is at least partly because the expressed SpCas9 may complex with the sgRNA designed for SpdCas9‐mediated suppression and lead to off‐target mutagenesis.…”

“…However, virtually all previous studies exploiting CRISPR or CRISPRi for E. coli engineering have used SpCas9 (Ding, Weng, Du, Chen, & Kang, 2017;Heo et al, 2017;Y. Jiang et al, 2015;Kiani et al, 2015;Liang et al, 2017;Liu et al;Pyne, Moo-Young, Chung, & Chou, 2015;Reisch & Prather, 2015;Ronda, Pedersen, Sommer, & Nielsen, 2016;Umenhoffer et al, 2017;Zhao et al, 2016) or SpdCas9 (Cress et al, 2015(Cress et al, , 2016Kim et al, 2016;S. Li, Jendresen, et al, 2016;Lv et al, 2015;Rath, Amlinger, Hoekzema, Devulapally, & Lundgren, 2015;Wu et al, 2015).…”

Antibiotic usage promotes the evolution of resistance against gepotidacin, a novel multi-targeting drug

“…To assess the phenotype of gepotidacin resistance-associated mutations, we individually reconstructed both GyrA D82N and ParC D79N mutations in E. coli K-12 MG1655. We utilized a previously described CRISPR-MAGE protocol to integrate mutations and counter select against the wild-type genotype 83,84 . Briefly, cell-containing pORTMAGE2 (Addgene #72677) were transformed with pCas9 (Addgene #42876) and were grown in LB + 100 µg/ml ampicillin + 20 µg/ml chloramphenicol broth at 30 o C. Next, pCRISPR plasmids (Addgene #42875) were constructed with crRNA sequences targeting the wild-type gyrA and parC loci in the vicinity of D82 and D79 according to a previously described protocol (oligos: ECGACRF1, ECGACRR1, and ECPCCRF1, ECPCCRR1, respectively).…”

CRISPR-interference based modulation of mobile genetic elements in bacteria

“…Therefore, genetic stabilization of bacterial hosts must include the complete elimination of their mobility. Based on this concept, the deletion of all transposable elements from the genomes of various E. coli strains (23)(24)(25) and Acinetobacter baylyi (26) have already proven to be a successful strategy to increase the stability of chromosomal or plasmid-based synthetic constructs. One of these endeavors, which removed all mobile DNA (prophages, cryptic prophages, ISes) from the chromosome of E. coli MG1655 by subsequently executing 42 precise deletions, took several years to accomplish in our laboratory (23).…”

“…One of these endeavors, which removed all mobile DNA (prophages, cryptic prophages, ISes) from the chromosome of E. coli MG1655 by subsequently executing 42 precise deletions, took several years to accomplish in our laboratory (23). Later, the availability of programmable CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) (27,28) endonucleases revolutionized the genome-wide inactivation of mobile DNA both in bacteria (24) and in mammalian cells (29,30). Furthermore, an in vivo targeted base-editing technique has been recently developed that could potentially inactivate mobile DNA elements in E. coli using a CRISPR/Cas-targeted cytidine deaminase (31).…”