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

ABSTRACT Considered a serious threat by the Centers for Disease Control and Prevention, multidrug-resistant Enterococcus faecium is an increasing cause of hospital-acquired infection. Here, we provide details on a single-plasmid CRISPR-Cas12a system for generating clean deletions and insertions. Single manipulations were carried out in under 2 weeks, with successful deletions/insertions present in >80% of the clones tested. Using this method, we generated three individual clean deletion mutations in the acpH, treA, and lacL genes and inserted codon-optimized unaG, enabling green fluorescent protein (GFP)-like fluorescence under the control of the trehalase operon. The use of in vivo recombination for plasmid construction kept costs to a minimum. IMPORTANCE 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. Here, we show that CRISPR-Cas12a can be used to quickly (under 2 weeks) and cheaply delete or insert genes into the E. faecium genome. This substantial improvement over current methods should speed up research on this important opportunistic pathogen.


Figure S1
Map of pJC005 & pUC19.pcRNA. The specific SOE PCR product to generate a gene specific knockout/in is inserted following linearization of pJC005 via BtgZI restriction digestion or PCR with oligos oJC218-oJC219. pUC19.pcRNA acts as template DNA for the small RNA promoter and CRISPR Spacer. The CRISPR Spacer is unique to each knockout construct and added to the reverse oligo.

Construction of Plasmids
PCR products were amplified using the Q5 DNA polymerase according to the manufacturer's protocol. Plasmid and genomic DNA concentrations for PCR ranged from 1-10 ng and 150-400 ng, respectively. Primers used to amplify the inserts contained overhangs to allow for in vivo assembly. PCR products from plasmid DNA were DpnI-digested to remove template DNA and minimize transformation background. The Zymo DNA Clean & Concentrator TM -5 was used to clean all PCR products before use.
To knock out specific genes, pJC005 requires three inserts: a small RNA promoter driving the CRISPR protospacer RNA and up and downstream arms homologous to the flanking regions of the target gene. To act as template DNA, we synthesized a 397 bp small RNA promoter (from Clostridium beijerinckii) and nonsense spacer sequence between two 19 bp repeats and cloned the sequence into pUC19 following EcoRI-XhoI digestion generating pUC19.pcRNA.

GCTGGAATTCGTCATAATCTTTAATTTGAAAAGATTTAAGGCTTATTTAAATAAAAAATATGAGGGAAGAATTGATA TAAATTTAATTTTGTTATTGTATTATGGTATGTATGGAATAAATTTAACATAAAGACAGTAATAATGTTCTTGAATT TAGACTTTTTATGTGTTATCATTAACAAGTATCAAAAATGACATTTAATAAATTAATAATAATTTTAAAAATATATT TTTGATAAAAGCAATGATTAACATGGTTTGACGTCTGAGAAGAGACGATTTTCTCAATAGGAGAAATTAAGGTGCAA acccttatcattccaccaTAATTTCTACTCTTGTAGATtcctcaaagagcatatggatatgAATTTCTACTCTTGTA GATCTCGAGGCC
Restriction sites -small RNA promoter -repeats The CRISPR protospacer is a 23 bp spacer sequence homologous to the target sequence with a protospacer adjacent motif (PAM) of 5`-TTTV-3` immediately upstream. The upstream and downstream sequences are used for homologous recombination and are between 500 and 800 bp.
To knockout the treA gene in E. faecium NCTC7171, pJC005.XtreA was generated. Primers oMC046/047 and oMC048/49 were used to amplify the upstream and downstream arms of pJC005.XtreA, respectively, from E. faecium NCTC7171 genomic DNA. Primer oJC221 is a universal primer that binds to the small RNA promoter template DNA and has overhangs homologous to linearized pJC005. Primer oMC045 binds to the small RNA promoter and repeat region, and its overhangs contain the specific treA target sequence.
To knockout the lacL gene, pJC005.XlacL was generated. Primers oMC056/057 and oMC058/59 were used to amplify the upstream and downstream arms, respectively. The small RNA promoter was amplified from pUC19.pcRNA using primers oJC221 and oMC055.
To knockout the acpH gene, pJC005.XacpH was generated. Primers oJC075/076 and oJC077/78 were used to amplify the upstream and downstream arms, respectively. The small RNA promoter was amplified from pUC19.pcRNA using primers oJC221 and oJC074. SOE PCR was used to ligate the small RNA promoter containing the CRISPR spacer and the up-anddownstream homologous arms. Individual PCR products were cleaned by column purification and 50 ng of the largest fragment and equimolar amounts of the smaller pieces were mixed in a 20 μl final volume Q5 PCR reaction. The NEB Tm calculator was used to determine annealing temperatures for the following cycling conditions: 98°C for 10 sec, then 10 cycles of 98°C for 10 sec, Tm of homologous regions for 30 sec, 72°C for 30 sec, and a final extension at 72°C for 10 min. Following the first round of SOE PCR, 1 μl of each universal SOE primer (oMC087-oMC088) was added, and the reaction returned to the thermocycler for 98°C for 2 min, then 15 cycles of 98°C for 10 sec, 65°C for 30 sec, 72°C for 30 sec; and a final extension at 72°C for 10 min. One μl of the SOE PCR product was used directly for in vivo cloning if a single band was observed following gel electrophoresis. If multiple bands were present, the correct size band was gel extracted before use.
The knockout plasmids for this study were assembled in vivo by High Efficiency NEB® 10-beta Competent E. coli. Prior to transformation, 2 µl the SOE PCR product and 50 ng of linearized pJC005 backbone were mixed and added to 50 µl of chemically competent E. coli DB10 cells. Cells were incubated on ice for 30 minutes, heat shocked at 42°C for 30 seconds and incubated on ice for another 3 minutes.
After addition of 450 µl SOC, the cells were incubated at 37°C with shaking for 1 hour. Transformed cells were selected for on LB agar supplemented with 200 µg/ml erythromycin. Transformants with the correct plasmid were confirmed with primers oJC070 and oJC071.
To knock-in the unaG gene, pJC005.XtreA::unaG was generated to replace the treA gene with the unaG gene. pJC005.XtreA was linearized in between the treA upstream and downstream arms using primers oMC099 and oMC100, both of which contain overhangs for the unaG gene. The codon optimized unaG gene was amplified using primers oMC101 and oMC102 from pMC001 (unaG gene under TetR control, this study). Primers oMC101 and oMC102 have overhangs homologous to the treA upstream and downstream arms, respectively. pJC005.treA::unaG was also assembled in vivo by High Efficiency NEB® 10-beta Competent E. coli. The transformation protocol resembled the knockout plasmid protocol, and the transformants with the correct plasmid were confirmed with primers oJC070 and oJC071.
Primers used. CRISPR Spacer targets are underlined.