Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a

Chua, Michelle J.; Collins, James W.

doi:10.1128/spectrum.02427-21

Cited by 9 publications

(4 citation statements)

References 31 publications

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“…Although CRISPR-Cas has been used for genome editing in a variety of microorganisms, the generation of escapers is a ubiquitous phenomenon. ,,,,,− Escapers in CRISPR-Cas limit its applications such as for new antimicrobials based on CRISPR-Cas, though the delivery of the CRISPR-Cas is another bottleneck. In this study, we focused on the CRISPR-Cas9-based gene editing system pEcCas/pEcgRNA established in our previous research, which showed that E. coli MG1655 can survive the DBS generated by Cas9 with a rate of 10 –5 –10 –3 , which is much higher than the acceptable rate (10 –8 ) recommended by the NIH (Figure ).…”

Section: Discussionmentioning

confidence: 99%

Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism

Sun

et al. 2023

ACS Synth. Biol.

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Due to the high specificity in targeting DNA and highly convenient programmability, CRISPR-Cas-based antimicrobials applied for eliminating specific strains such as antibiotic-resistant bacteria in the microbiome were gradually developed. However, the generation of escapers makes the elimination efficiency far lower than the acceptable rate (10–8) recommended by the National Institutes of Health. Here, a systematic study was carried out providing insight into the escaping mechanisms in Escherichia coli, and strategies for reducing the escapers were devised accordingly. We first showed an escape rate of 10–5–10–3 in E. coli MG1655 under the editing of pEcCas/pEcgRNA established previously. Detailed analysis of the escapers obtained at ligA site in E. coli MG1655 uncovered that the disruption of cas9 was the main cause of the generation of survivors, notably the frequent insertion of IS5. Hence, the sgRNA was next designed to target the “perpetrator” IS5, and subsequently the killing efficiency was improved 4-fold. Additionally, the escape rate in IS-free E. coli MDS42 was also tested at the ligA site, ∼10-fold decrease compared with MG1655, but the disruption of cas9 was still observed in all survivors manifested in the form of frameshifts or point mutations. Thus, we optimized the tool itself by increasing the copy number of cas9 to retain some cas9 that still has the correct DNA sequence. Fortunately, the escape rates dropped below 10–8 at 9 of the 16 tested genes. Furthermore, the λ-Red recombination system was added to generate the pEcCas-2.0, and a 100% gene deletion efficiency was achieved at genes cadA, maeB, and gntT in MG1655, whereas those genes were edited with low efficiency previously. Last, the application of pEcCas-2.0 was then expanded to the E. coli B strain BL21(DE3) and W strain ATCC9637. This study reveals the mechanism of E. coli surviving Cas9-mediated death, and a highly efficient editing tool is established based on the mechanism, which will accelerate the further application of CRISPR-Cas.

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Section: Discussionmentioning

confidence: 99%

Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism

Sun

et al. 2023

ACS Synth. Biol.

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“…A CRISPR-Cas12 system was used to generate clean deletion mutants of ptsHI in E. faecium NCTC7171 as previously described (Chua and Collins 2022 ). Briefly, three inserts, a small RNA promoter driving the CRISPR protospacer RNA, and up- and downstream arms homologous to the flanking regions surrounding the ptsHI were amplified by PCR with primers containing splicing by overlap extension (SOE) PCR-compatible overhang regions (5′- -3′ and 5′- -3′; 5′- -3′ and 5′- -3′; and 5′- -3′ and 5′- -3′, respectively).…”

Section: Methodsmentioning

confidence: 99%

The role of the universal sugar transport system components PtsI (EI) and PtsH (HPr) in Enterococcus faecium

Hallenbeck,

Chua,

Collins

2024

FEMS Microbes

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Vancomycin-resistant enterococci (VRE) pose a serious threat to public health because of their limited treatment options. Therefore, there is an increasing need to identify novel targets to develop new drugs. Here, we examined the roles of the universal PTS components, PtsI and PtsH, in Enterococcus faecium to determine their roles in carbon metabolism, biofilm formation, stress response, and the ability to compete in the gastrointestinal tract. Clean deletion of ptsHI resulted in a significant reduction in the ability to import and metabolize simple sugars, attenuated growth rate, reduced biofilm formation, and decreased competitive fitness both in vitro and in vivo. However, no significant difference in stress survival was observed when compared with the wild type. These results suggest that targeting universal or specific PTS may provide a novel treatment strategy by reducing the fitness of E. faecium.

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“…Recently, CRISPR/Cas12a has been proved to be able to simultaneously edit pyrG, pksP , and kusA genes of Aspergillus aculeatus TBRC 277 (an industrially related cell factory), with an efficiency of up to 40% [ 59 ]. In conclusion, CRISPR/Cas12-mediated microbial genome editing mainly involves in strain improvement, such as the production of valuable steroidal pharmaceuticals [ 58 ] and bioproducts [ 59 ], as well as high-performance chassis [ 60 ], and fundamental research, for instance, the study of plant-fungal interactions [ 61 , 62 ] and the pathogenesis of important opportunistic pathogens [ 63 ] (Table 4 ). For ease of understanding, based on existing reports, CRISPR/Cas12-mediated genome editing was represented in Fig.…”

Section: Crispr/cas12 System Involves In Genome Editing Of Microorgan...mentioning

confidence: 99%

Research progress on nucleic acid detection and genome editing of CRISPR/Cas12 system

et al. 2023

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Purpose This work characterizes the applications of CRISPR/Cas12 system, including nucleic acid detection, animal, plant and microbial genome editing. Methods The literature on CRISPR/Cas12 system was collected and reviewed. Results CRISPR/Cas system is an acquired immune system derived from bacteria and archaea, which has become the most popular technology around the world because of its outstanding contribution in genome editing. Type V CRISPR/Cas systems are distinguished by a single RNA-guided RuvC nuclease domain with single effector molecule. Cas12a, the first reported type V CRISPR/Cas system, targets double-stranded DNA (dsDNA) adjacent to PAM sequences and trans-cleaves single-stranded DNA (ssDNA). We present the applications of CRISPR/Cas12 system for nucleic acid detection and genome editing in animals, plants and microorganisms. Furthermore, this review also summarizes the applications of other Cas12 proteins, such as Cas12b, Cas12c, Cas12d, and so on, which further widen the application prospects of CRISPR/Cas12 system. Conclusions Knowledge of the applications of CRISPR/Cas12 system is necessary for improving the understanding of the functional diversity of CRISPR/Cas12 system and also provides significant references for further research and utilization of CRISPR/Cas12 in other new fields. Graphical abstract

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Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a

Abstract: Enterococcus faecium is increasingly associated with hard-to-treat antibiotic-resistant infections. The ability to generate clean genomic alterations is the first step in generating a complete mechanistic understanding of how E. faecium acquires pathogenic traits and causes disease.

Cited by 9 publications

References 31 publications

Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism

Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism

The role of the universal sugar transport system components PtsI (EI) and PtsH (HPr) in Enterococcus faecium

Research progress on nucleic acid detection and genome editing of CRISPR/Cas12 system

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