Avoiding CRISPR: Plasmid design for genetic engineering

Clustered regularly interspersed short palindromic repeats (CRISPR) is a natural defense system for bacteria and archaea against foreign DNA and RNA. Specifically, short snippets of foreign DNA or RNA are incorporated into protospacer adjacent motif (PAM) repeats sequences in the genome of the bacterial species, and serve as molecular memory of past infections by viruses. These repeats are transcribed by RNA polymerases and perform constant surveillance of the bacterial cell cytoplasm for foreign DNA. Once detected, PAM sequences would bind to the foreign DNA leading to the recruitment of Cas endonuclease protein that cut the foreign DNA. Plasmids are double stranded DNA vectors that serve to carry foreign genes into the cell for genetic engineering. Hence, plasmids are also foreign DNA with respect to the CRISPR system of the cell. To avoid destruction by the Cas protein, plasmid should not contain sequences that would bind to any of the PAM sequences encoded in the genome of the bacterial species. Thus, the PAM sequences of each bacterial species where genetic engineering is to be performed should be sequenced, and the knowledge gained utilized in the design of plasmid vectors that do not carry any of the sequences encoded by PAM repeats. Such an approach would help reduce the chances of destruction of plasmid vector once it was introduced to the cell, and would help improve the efficiency of plasmid transduction and genetic engineering. Collectively, CRISPR is a natural cellular defense system that could destroy introduced plasmid vector through recognition by PAM repeat sequences encoded in the cell’s genome. Sequencing of the PAM sequence of the bacterial species followed by careful design of the plasmid DNA sequence would significantly reduce the chances of destruction of the vector by CRISPR once it was introduced into the cell.

Avoiding CRISPR: Plasmid design for genetic engineering

Clustered regularly interspersed short palindromic repeats (CRISPR) is a natural defense system for bacteria and archaea against foreign DNA and RNA. Specifically, short snippets of foreign DNA or RNA are incorporated into protospacer adjacent motif (PAM) repeats sequences in the genome of the bacterial species, and serve as molecular memory of past infections by viruses. These repeats are transcribed by RNA polymerases and perform constant surveillance of the bacterial cell cytoplasm for foreign DNA. Once detected, PAM sequences would bind to the foreign DNA leading to the recruitment of Cas endonuclease protein that cut the foreign DNA. Plasmids are double stranded DNA vectors that serve to carry foreign genes into the cell for genetic engineering. Hence, plasmids are also foreign DNA with respect to the CRISPR system of the cell. To avoid destruction by the Cas protein, plasmid should not contain sequences that would bind to any of the PAM sequences encoded in the genome of the bacterial species. Thus, the PAM sequences of each bacterial species where genetic engineering is to be performed should be sequenced, and the knowledge gained utilized in the design of plasmid vectors that do not carry any of the sequences encoded by PAM repeats. Such an approach would help reduce the chances of destruction of plasmid vector once it was introduced to the cell, and would help improve the efficiency of plasmid transduction and genetic engineering. Collectively, CRISPR is a natural cellular defense system that could destroy introduced plasmid vector through recognition by PAM repeat sequences encoded in the cell’s genome. Sequencing of the PAM sequence of the bacterial species followed by careful design of the plasmid DNA sequence would significantly reduce the chances of destruction of the vector by CRISPR once it was introduced into the cell.

Plasmid Transduction Using Bacteriophage Φadh for Expression of CC Chemokines by Lactobacillus gasseri ADH

ABSTRACT Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Φadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Φadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.

Identification and Characterization of Lactococcal-Prophage-Carried Superinfection Exclusion Genes

ABSTRACT Superinfection exclusion (Sie) proteins are prophage-encoded phage resistance systems. In this study, genes encoding Sie systems were identified on the genomes of Lactococcus lactis subsp. cremoris MG1363 and L. lactis subsp. lactis IL1403. These Sie systems are genetically distinct and yet were shown to act specifically against a particular subset of the 936 phage group. Each of the systems allows normal phage adsorption while affecting plasmid transduction and intracellular phage DNA replication, which points to the blocking of phage DNA injection as their common mode of action. Sie-specifying genes found on the MG1363 prophages are also present in various lactococcal strains, whereas the prophage-encoded Sie systems of IL1403 do not appear to be as widely disseminated.

Plasmid Transfer via Transduction from Streptococcus thermophilus to Lactococcus lactis

ABSTRACT Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.

Gene Transfer by Transduction in the Marine Environment

ABSTRACT To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 × 10−7 to 5.13 × 10−9transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with thenptII probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10−8 to 3.7 × 10−8transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 1014transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.

Role of Erf recombinase in P22-mediated plasmid transduction.

In the absence of host RecA function, plasmid transduction by bacteriophage P22 can be mediated by Erf recombinase. Erf is not carried on the infecting particle but synthesized upon infection. In the recipient cell, Erf can promote both generalized plasmid transduction (which requires the circularization of plasmids transduced as linear multimers) and specialized plasmid transduction (which requires the release of plasmid DNA from linear plasmid-phage cointegrates). Both processes of Erf-mediated plasmid transduction require host RecBCD function. In contrast, RecBCD is not required for Erf-mediated circularization of P22 DNA.