Genome editing in bacteria encompasses a wide array of laborious and multi-step methods such as suicide plasmids. The discovery and applications of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas based technologies have revolutionized genome editing in eukaryotic organisms due to its simplicity and programmability. Nevertheless, this system has not been as widely favored for bacterial genome editing. In this review, we summarize the main approaches and difficulties associated with CRISPR-Cas-mediated genome editing in bacteria and present some alternatives to circumvent these issues, including CRISPR nickases, Cas12a, base editors, CRISPR-associated transposases, prime-editing, endogenous CRISPR systems, and the use of pre-made ribonucleoprotein complexes of Cas proteins and guide RNAs. Finally, we also address fluorescent-protein-based methods to evaluate the efficacy of CRISPR-based systems for genome editing in bacteria. CRISPR-Cas still holds promise as a generalized genome-editing tool in bacteria and is developing further optimization for an expanded application in these organisms. This review provides a rarely offered comprehensive view of genome editing. It also aims to familiarize the microbiology community with an ever-growing genome-editing toolbox for bacteria.

Significance: ATP, the fuel of life, is produced in living cells by a complex molecular machine consisting of two motors linked by a rotor. One motor generates rotation by consuming energy derived from oxidative metabolism or photosynthesis; the other uses energy transmitted by the rotor to put ATP molecules together from their building blocks, ADP and phosphate. One such intact machine from the α-proteobacterium Paracoccus denitrificans has been induced to form crystals, providing the means of deducing a blueprint of the machine, giving details of how its components are organized, and providing insights into how it works. The mechanistic principles deduced from the bacterial machine apply to similar molecular machines found in all living organisms.

The F ₁ F _O and F ₁ ‐ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome‐reconstituted holoenzyme preserves its tight coupling between F ₁ and F _O sectors, as evidenced by its high sensitivity to the F _O inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N‐terminal sequencing of the band profile in SDS‐PAGE of the F ₁ and F ₁ F _O preparations showed a novel 11‐kDa protein in addition to the 5 canonical α, β, γ, δ, and ε subunits present in all known F ₁ ‐ATPase complexes. BN‐PAGE followed by 2D‐SDS‐PAGE confirmed the presence of this 11‐kDa protein bound to the native F ₁ F _O ‐ATP synthase of P. denitrificans , as it was observed after being isolated. The recombinant 11 kDa and ε subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F ₁ F _O and soluble F ₁ ‐ATPase complexes. The 11‐kDa protein, but not the ε subunit, inhibited the F ₁ F _O and F ₁ ‐ATPase activities of P. denitrificans . The 11‐kDa protein was also found in Rhodobacter sphaeroides associated to its native F ₁ F _O ‐ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11‐kDa protein on the F ₁ F _O ‐ATPase nanomotor of P. denitrificans and closely related α‐proteobacteria.—Morales‐Ríos, E., de la Rosa‐Morales, F., Mendoza‐Hernández, G., Rodríguez‐Zavala, J. S., Celis, H., Zarco‐Zavala, M., García‐Trejo, J. J. A novel 11‐kDa inhibitory subunit in the F ₁ F _O ATP synthase of Paracoccus denitrificans and related α‐proteobacteria. FASEB J . 24, 599–608 (2010). www.fasebj.org