staphylococci. Microbiology 159:421–435.
https://doi.org/10.1099/mic.0.061705-0
7. Monk IR, Shah IM, Xu M et al (2012) Transforming the untransformable: application of
direct transformation to manipulate genetically
Staphylococcus aureus and Staphylococcus epidermidis. mBio 3(2). https://doi.org/10.
1128/mBio.00277-11
8. Liu Q, Jiang Y, Shao L et al (2017) CRISPR/
Cas9-based efficient genome editing in Staphylococcus aureus. Acta Biochim Biophys Sin
49:764–770.
https://doi.org/10.1093/
abbs/gmx074
9. Datta S, Costantino N, Zhou X, Court DL
(2008) Identification and analysis of recombineering functions from gram-negative and
gram-positive bacteria and their phages. Proc
Natl Acad Sci U S A 105:1626–1631. https://
doi.org/10.1073/pnas.0709089105
10. Selle K, Barrangou R (2015) Harnessing
CRISPR-Cas systems for bacterial genome
editing. Trends Microbiol 23:225–232.
https://doi.org/10.1016/j.tim.2015.01.008
11. Penewit K, Holmes EA, McLean K et al (2018)
Efficient and scalable precision genome editing
in Staphylococcus aureus through conditional
recombineering and CRISPR/Cas9-mediated
counterselection. mBio 9(1). https://doi.
org/10.1128/mBio.00067-18
12. Ellis HM, Yu D, DiTizio T, Court DL (2001)
High efficiency mutagenesis, repair, and engineering of chromosomal DNA using singlestranded oligonucleotides. Proc Natl Acad Sci
U S A 98:6742–6746. https://doi.org/10.
1073/pnas.121164898
13. Barrangou R, van Pijkeren J-P (2016) Exploiting CRISPR-Cas immune systems for genome
editing in bacteria. Curr Opin Biotechnol
37:61–68. https://doi.org/10.1016/j.copbio.
2015.10.003
14. Reisch CR, Prather KLJ (2015) The no-SCAR
(scarless Cas9 assisted recombineering) system
for genome editing in Escherichia coli. Sci Rep
5:15096. https://doi.org/10.1038/srep15096
15. Jiang W, Bikard D, Cox D et al (2013)
RNA-guided editing of bacterial genomes
using CRISPR-Cas systems. Nat Biotechnol
31:233–239. https://doi.org/10.1038/nbt.
2508
16. Charpentier E, Anton AI, Barry P et al (2004)
Novel cassette-based shuttle vector system for
gram-positive bacteria. Appl Environ Microbiol 70:6076–6085. https://doi.org/10.
1128/AEM.70.10.6076-6085.2004
17. Sau S, Sun J, Lee CY (1997) Molecular characterization and transcriptional analysis of type
8 capsule genes in Staphylococcus aureus. J
Bacteriol 179:1614–1621
18. Huang H, Zheng G, Jiang W et al (2015)
One-step high-efficiency CRISPR/Cas9mediated genome editing in Streptomyces.
Acta Biochim Biophys Sin 47:231–243.
https://doi.org/10.1093/abbs/gmv007
19. Altenbuchner J (2016) Editing of the Bacillus
subtilis genome by the CRISPR-Cas9 system.
Appl Environ Microbiol 82(17):5421–5427.
https://doi.org/10.1128/AEM.01453-16
20. Mougiakos I, Bosma EF, de Vos WM et al
(2016) Next generation prokaryotic engineering: the CRISPR-Cas toolkit. Trends Biotechnol 34:575–587. https://doi.org/10.1016/j.
tibtech.2016.02.004
21. Xu T, Li Y, Shi Z et al (2015) Efficient genome
editing in Clostridium cellulolyticum via
CRISPR-Cas9 nickase. Appl Environ Microbiol 81:4423–4431. https://doi.org/10.
1128/AEM.00873-15
22. van der Vossen JM, van der Lelie D, Venema G
(1987) Isolation and characterization of Streptococcus cremoris Wg2-specific promoters.
Appl Environ Microbiol 53:2452–2457
23. Qi LS, Larson MH, Gilbert LA et al (2013)
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene
expression. Cell 152:1173–1183. https://doi.
org/10.1016/j.cell.2013.02.022
24. Monk IR, Tree JJ, Howden BP et al (2015)
Complete bypass of restriction systems for
major Staphylococcus aureus lineages. mBio
6:e00308-00315. https://doi.org/10.1128/
mBio.00308-15
25. Schenk S, Laddaga RA (1992) Improved
method for electroporation of Staphylococcus
aureus. FEMS Microbiol Lett 73:133–138
26. Moreno-Mateos MA, Vejnar CE, Beaudoin
J-D et al (2015) CRISPRscan: designing highly
efficient sgRNAs for CRISPR-Cas9 targeting
in vivo. Nat Methods 12:982–988. https://
doi.org/10.1038/nmeth.3543
27. Mosberg JA, Gregg CJ, Lajoie MJ et al (2012)
Improving lambda red genome engineering in
Escherichia coli via rational removal of endogenous nucleases. PLoS One 7:e44638. https://
doi.org/10.1371/journal.pone.0044638
28. Wang HH, Isaacs FJ, Carr PA et al (2009)
Programming cells by multiplex genome engineering and accelerated evolution. Nature
460:894–898.
https://doi.org/10.1038/
nature08187
29. Sawitzke JA, Costantino N, Li X-T et al (2011)
Probing cellular processes with oligo-mediated
recombination and using the knowledge
gained to optimize recombineering. J Mol
142
Kelsi Penewit and Stephen J. Salipante
https://doi.org/10.1099/mic.0.061705-0
7. Monk IR, Shah IM, Xu M et al (2012) Transforming the untransformable: application of
direct transformation to manipulate genetically
Staphylococcus aureus and Staphylococcus epidermidis. mBio 3(2). https://doi.org/10.
1128/mBio.00277-11
8. Liu Q, Jiang Y, Shao L et al (2017) CRISPR/
Cas9-based efficient genome editing in Staphylococcus aureus. Acta Biochim Biophys Sin
49:764–770.
https://doi.org/10.1093/
abbs/gmx074
9. Datta S, Costantino N, Zhou X, Court DL
(2008) Identification and analysis of recombineering functions from gram-negative and
gram-positive bacteria and their phages. Proc
Natl Acad Sci U S A 105:1626–1631. https://
doi.org/10.1073/pnas.0709089105
10. Selle K, Barrangou R (2015) Harnessing
CRISPR-Cas systems for bacterial genome
editing. Trends Microbiol 23:225–232.
https://doi.org/10.1016/j.tim.2015.01.008
11. Penewit K, Holmes EA, McLean K et al (2018)
Efficient and scalable precision genome editing
in Staphylococcus aureus through conditional
recombineering and CRISPR/Cas9-mediated
counterselection. mBio 9(1). https://doi.
org/10.1128/mBio.00067-18
12. Ellis HM, Yu D, DiTizio T, Court DL (2001)
High efficiency mutagenesis, repair, and engineering of chromosomal DNA using singlestranded oligonucleotides. Proc Natl Acad Sci
U S A 98:6742–6746. https://doi.org/10.
1073/pnas.121164898
13. Barrangou R, van Pijkeren J-P (2016) Exploiting CRISPR-Cas immune systems for genome
editing in bacteria. Curr Opin Biotechnol
37:61–68. https://doi.org/10.1016/j.copbio.
2015.10.003
14. Reisch CR, Prather KLJ (2015) The no-SCAR
(scarless Cas9 assisted recombineering) system
for genome editing in Escherichia coli. Sci Rep
5:15096. https://doi.org/10.1038/srep15096
15. Jiang W, Bikard D, Cox D et al (2013)
RNA-guided editing of bacterial genomes
using CRISPR-Cas systems. Nat Biotechnol
31:233–239. https://doi.org/10.1038/nbt.
2508
16. Charpentier E, Anton AI, Barry P et al (2004)
Novel cassette-based shuttle vector system for
gram-positive bacteria. Appl Environ Microbiol 70:6076–6085. https://doi.org/10.
1128/AEM.70.10.6076-6085.2004
17. Sau S, Sun J, Lee CY (1997) Molecular characterization and transcriptional analysis of type
8 capsule genes in Staphylococcus aureus. J
Bacteriol 179:1614–1621
18. Huang H, Zheng G, Jiang W et al (2015)
One-step high-efficiency CRISPR/Cas9mediated genome editing in Streptomyces.
Acta Biochim Biophys Sin 47:231–243.
https://doi.org/10.1093/abbs/gmv007
19. Altenbuchner J (2016) Editing of the Bacillus
subtilis genome by the CRISPR-Cas9 system.
Appl Environ Microbiol 82(17):5421–5427.
https://doi.org/10.1128/AEM.01453-16
20. Mougiakos I, Bosma EF, de Vos WM et al
(2016) Next generation prokaryotic engineering: the CRISPR-Cas toolkit. Trends Biotechnol 34:575–587. https://doi.org/10.1016/j.
tibtech.2016.02.004
21. Xu T, Li Y, Shi Z et al (2015) Efficient genome
editing in Clostridium cellulolyticum via
CRISPR-Cas9 nickase. Appl Environ Microbiol 81:4423–4431. https://doi.org/10.
1128/AEM.00873-15
22. van der Vossen JM, van der Lelie D, Venema G
(1987) Isolation and characterization of Streptococcus cremoris Wg2-specific promoters.
Appl Environ Microbiol 53:2452–2457
23. Qi LS, Larson MH, Gilbert LA et al (2013)
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene
expression. Cell 152:1173–1183. https://doi.
org/10.1016/j.cell.2013.02.022
24. Monk IR, Tree JJ, Howden BP et al (2015)
Complete bypass of restriction systems for
major Staphylococcus aureus lineages. mBio
6:e00308-00315. https://doi.org/10.1128/
mBio.00308-15
25. Schenk S, Laddaga RA (1992) Improved
method for electroporation of Staphylococcus
aureus. FEMS Microbiol Lett 73:133–138
26. Moreno-Mateos MA, Vejnar CE, Beaudoin
J-D et al (2015) CRISPRscan: designing highly
efficient sgRNAs for CRISPR-Cas9 targeting
in vivo. Nat Methods 12:982–988. https://
doi.org/10.1038/nmeth.3543
27. Mosberg JA, Gregg CJ, Lajoie MJ et al (2012)
Improving lambda red genome engineering in
Escherichia coli via rational removal of endogenous nucleases. PLoS One 7:e44638. https://
doi.org/10.1371/journal.pone.0044638
28. Wang HH, Isaacs FJ, Carr PA et al (2009)
Programming cells by multiplex genome engineering and accelerated evolution. Nature
460:894–898.
https://doi.org/10.1038/
nature08187
29. Sawitzke JA, Costantino N, Li X-T et al (2011)
Probing cellular processes with oligo-mediated
recombination and using the knowledge
gained to optimize recombineering. J Mol
142
Kelsi Penewit and Stephen J. Salipante