Genome Editing in Bacteria: CRISPR-Cas and Beyond

Abstract

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.

Keywords: CRISPR-Cas; genome editing; prokaryotes; ribonucleoprotein; suicide plasmids.

Figure 1

The three stages of the CRISPR-Cas (type II) bacterial adaptive immune system. During CRISPR adaptation, the injection of phage DNA into bacterial cell activates the adaptation module proteins which excise spacer-sized fragments of phage DNA for incorporation into CRISPR loci. During CRISPR RNA biogenesis, CRISPR loci are transcribed and resulting pre-crRNA is processed by a Cas9/RNaseIII complex at repeat sequences to generate mature crRNAs that couple to tracrRNA (gRNA). Individual gRNAs are bound by Cas protein effectors (e.g., Cas9). After a new phage infection with sequences matching a CRISPR spacer appears in the cell (lower right), specific Cas/gRNA complexes bind to viral DNA and cleave it.

Figure 2

Standard methods for genome editing in bacteria. Suicide plasmids. (A) The classic approach consists in transforming with a non-replicating plasmid (usually with a transposon element, e.g., mob), which harbors a mutated recombination template and an antibiotic resistance marker (ABr). Antibiotic treatment will select only colonies that undergo homologous recombination to incorporate the plasmid sequence (including the mutant gene) at the target locus (disrupting gene X). (B) In the “scarless” variant a SceI site is incorporated in the plasmid to be transformed in a I-SceI expressing strain under an inducible promoter (pTet). After a first round of antibiotic treatment, cointegrating colonies harboring the plasmid sequence and the wild-type allele at the target gene locus are selected. Addition of chlortetracycline (CTc) induces I-SceI expression to cleave the target locus, which enhances homologous recombination to eliminate plasmid sequence resulting in either, reversion to wild-type or fixation of the mutant allele. (C) Recombineering (lambda red system) for targeted gene disruption. A targeting construct with 50 nt of homologous sequence at the 5′ and 3′ ends and antibiotic resistance marker is made by PCR. PCR template is electroporated and expression of the lambda Red proteins is induced (Ex. Heat shock at 42 °C). Gam inhibits RecBCD nuclease activity upon linear DNA (protecting the targeting construct). Exo generates 3′ overhangs in the DNA linear template, which are accessed by bet protein to facilitate homologous recombination and integration and disruption of the target gene (gene x). Edited colonies are then selected by antibiotic treatment. (D) ClosTron method. A type II intron with transposon activity is cloned within a disrupted antibiotic resistance cassette in a plasmid. After transformation, the intron, which has been modified with a specific, homologous sequence, targets the gene of interest (G.O.I) and disrupts it leaving behind a plasmid with a functional antibiotic resistance marker. Antibiotic selection then enhances and simplifies the obtention of mutant colonies.

Figure 3

Strategies used for CRISPR-Cas based genome editing in bacteria. (A) Editing via homologous recombination: Recombineering with a linear DNA template is followed by counterselection with CRISPR nucleases. A heterologous recombinase (e.g., λ red, RecT) is introduced via a plasmid (or phage) into the cell and co-transformed with the linear DNA template and CRISPR-nuclease plasmid with respective antibiotic-resistance marker (ABr). Genome editing may also be directed with a plasmid-encoded recombination template (RT) and endogenous or heterologous recombinase. The recombination template can be placed on the same plasmid encoding the CRISPR machinery for an all-in-one plasmid system, or it can be placed on a separate plasmid before transforming the CRISPR nuclease/gRNA plasmid. One-plasmid system is more streamlined, but due to its larger size it can be hard to transform, and cloning may not be possible if the gRNA can target the genome of the cloning strain. (B) Editing via the non-homologous end-joining (NHEJ) pathway. Depending on the strain, ku and/or ligD can be encoded on the CRISPR nuclease/gRNA plasmid and transformed into the strain. (C) Alternative end joining (A-EJ) pathway can be found natively in many bacterial species with incomplete NHEJ. It does not require the introduction of foreign Ku or LigD, and instead relies in microhomology-directed repair via RecBCD, nucleases, and LigA, leading to deletions of variable sizes (depending on the location of microhomologies) at the Cas9 cut site. For a more detailed insight on NHEJ and A-EJ mechanisms, the reader is advised to read [45]. All strategies require plasmid curing after nuclease targeting to isolate the mutant strain in order to avoid interference in pursuing downstream applications.

Figure 4

Molecular mechanism of SpCas9/gRNA cleavage of target DNA. (A) The main domains of Cas9 are illustrated next to a gRNA/target DNA secondary structure scheme. Adapted from [52]. (B) The first step is the PAM binding and phosphate lock loop binding, followed by DNA unwinding and finally the DNA recognition by gRNA and the target DNA cleavage by the RuvC and HNH nuclease domains at both strands. Critical Cas9 residues for each step are illustrated. Adapted from [54].

Figure 5

Alternative strategies to circumvent SpCas9 cytotoxicity. (A) Use of inducible systems to express SpCas9. Via an inducible promoter, SpCas9 expression is strongly repressed without inducer present (square) and only induced after exponential culture so that enough cells can survive and perform the genome edit. (B) Using less toxic nucleases to achieve editing. nCas9, which only cleaves one strand of DNA, and Cas12a (PAM: TTTV, where V is A or C or G) can be less toxic than SpCas9. (C) SpCas9-derived base editors eliminate the double-stranded break requirement for genome editing. A translational fusion of nCas9 (nickase) or dCas9 (“dead”), a cytidine (e.g., APOBEC1 in BE3) or adenosine (e.g., TadA-EcTadA+ in ABE2) deaminase domain, and an uracil DNA glycosylase inhibitor (UGI) is introduced on a plasmid into the cell. Upon nuclease binding and DNA strand unwinding, cytidines (or adenines) on the non-target strand within a defined window adjacent to the PAM are rapidly converted to uracil (or inosines), which is then processed as thymidine (or guanines) by DNA polymerase. (D) ShCAST insertion mechanism. A Tn7-like transposon from Scytonema hoffmani encodes transposases (tnsB, tnsC, tniQ), a nuclease deficient type V CRISPR protein (Cas12k) and guide RNA. This complex is combined with a cargo gene flanked by LE and RE elements. ShCAST is directed to the target locus and integrates the cargo gene 60–66 bp downstream of the PAM sequence, generating and insertion of the cargo gene flanked by the SE and RE elements, and a duplicated (4 bp) insertion site.

Figure 6

Ribonucleoprotein (RNP) approaches for CRISPR-Cas mediated genome editing. (A) RNP electroporation: Recombinant CRISPR nuclease (e.g., Cas9) is combined with in vitro transcribed or synthetic sgRNA to form active Cas9/sgRNA RNP complexes. Electroporation is usually used to form temporary holes in the bacterial cell wall to co-transform the RNPs with a linear single- or double-stranded recombination template harboring the desired edit plus additional mutations at the PAM site to avoid Cas9/sgRNA targeting. Targeting to the desired locus occurs, DNA double-strand break is formed 2–3 bp upstream PAM sequence, which is repaired by double homologous recombination with the linear DNA template. Wild-type allele is replaced by the mutant allele, which is fixed in the target genome or plasmid. Cas9/sgRNA RNPs are maintained only transiently in the cell and are degraded shortly after gene edition. This method does not require the introduction of antibiotic resistance markers or plasmid curing; however, its efficiency would be highly dependent on the transformation amenability and recombination machinery of the bacterial strain. (B) Cationic polymer conjugation with Cas9/sgRNA. Recombinant Cas9 is covalently linked to a cationic polymer (bPEI) followed by incubation with sgRNA to form CRISPR nanometric complexes. Electrostatic interactions facilitate binding and incorporation of Cr-nanocomplex into thick-cell walled Gram-positive bacteria. In this example, sgRNA targets incorporated Cas9 to the mecA gene, responsible for methicillin and oxacillin (oxa) resistance in Staphylococcus aureus (MRSA). Counterselection of MRSA is efficiently achieved compared to the incubation with RNP alone or combined with the cationic lipid lipofectamine.

Figure 7

Structures of Cas9 orthologs reveal conserved and divergent features among CRISPR–Cas9 systems (subtypes IIa-IIb-IIc). (A) (Left) Crystal Structure of the SaCas9–sgRNA–target DNA complex (PDB ID 5CZZ): (right) base-specific contacts between CTD domain and PAM nucleotides (NNGR(A or G)R(A or G)T). (B) (Left) Structure of FnCas9–sgRNA–target DNA complex (PDB ID 5B2O); (right) PAM (NGG) recognition by arginine residues of FnCas9 CTD domain. (C) (Left) Structure of CjCas9–sgRNA–target DNA complex (PDB ID 5X2H); (right) base-specific contacts between the CTD domain and PAM nucleotides (NNNV(A or C or G)R(A or G)Y(C or T)M(A or C) in this structure; optimal in vivo PAM has been determined as NNNNRYAC by [107]). The HNH domain has been deleted for crystallization, the red circle indicates its expected position in the CjCas9 structure [110]. PDB structures were drawn with UCSF Chimera v.1.14.