Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9

Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into <i>E. coli</i> a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene <i>rnlB</i> produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications

Comparison of pollen germination rates and pollen tube lengths between the Lat52-GFP, Lat52-Ann5-GFP 1, Lat52-G26-GFP 1 and 2 and Lat52-G257-GFP 1 and 2 homozygous lines.

<p>For pollen germination rate, the pollen germinated for 3 h in the presence of 0.6 µM BFA. Six hundred pollen grains and tubes were counted. For pollen tube length, the pollen germinated normally for 2 h and then for 2 h in the presence of 0.6 µM BFA. Three hundred pollen tubes were measured. Values represent the means ± _SD. *P<0.05 and **P<0.01 by Student's <i>t</i> test.</p

Identification of the constructs concerning Ann5.

<p>(A) Schematic representations of the constructs concerning Ann5. (B) <i>Ann5</i>-specific primers were designed to identify the level of overexpression of the <i>Ann5</i> transcript by RT-PCR performed for 25 cycles. Total RNA was extracted from open flowers of the wild-type (WT), Lat52-GFP, Lat52-Ann5-GFP 1, 2 and 3 and Ann5Pro-Ann5 1, 2 and 3 homozygous lines. <i>EF4A</i> was used as the control. (C) Pollen grains from the WT, Lat52-GFP and Lat52-Ann5-GFP 1, 2 and 3 lines were observed by epi-fluorescence microscopy with a GFP filter. Homo, homozygous lines; Hetero, heterozygous lines. Half of the pollen grains in the heterozygous lines expressed Ann5-GFP, and the other half did not express Ann5-GFP. Bar  = 20 µm. (D) Densitometry analysis of the results presented in (C). Fluorescence intensities (arbitrary units) across the whole pollen grain were calculated. More than 50 pollen grains for each line were quantified. Values represent the means ± _SD. *P<0.05 and **P<0.01 by Student's <i>t</i> test.</p

Analysis of the cytoplasmic streaming velocity in pollen tubes of the overexpression lines.

<p>(A) The velocity of cytoplasmic streaming in pollen tubes from the overexpression lines Lat52-GFP, Lat52-Ann5-GFP 1 and 3, Lat52-G26-GFP 1 and Lat52-G257-GFP 1 under normal conditions. Pollen grains were cultured on germination medium for 4 h. Cytosolic particles exhibiting continuous movement were selected at random for velocity analysis using the Image J software. Values represent the means ± _SD (n = 25). (B) The relative velocity of cytoplasmic streaming in pollen tubes from the Lat52-GFP, Lat52-Ann5-GFP 1 and 3, Lat52-G26-GFP 1 and Lat52-G257-GFP 1 overexpression lines in response to 0.6 µM BFA. The pollen had germinated normally for 2 h, followed by 2 h in the presence of 0.6 µM BFA. The velocity of the particles of each individual line in normal conditions was normalized to 1. The relative velocity was displayed as the proportion over the control. Values represent the means ± _SD. *P<0.05 and **P<0.01 (n = 25) by Student's <i>t</i> test.</p

Ann5Pro-G26 and Ann5Pro-G257 only partially rescued the aborted pollen genotype in Lat52-Ann5UTRi plants.

<p>(A) The expression level of the <i>Ann5</i> transcript in Lat52-Ann5UTRi and individual complementation plants. Total RNA was isolated from the open flowers and used for RT-PCR. <i>EF4A</i> was used as the control. (B) Quantification of the sterile pollen grains from Lat52-Ann5UTRi and the individual complementation plants. Values represent the means ± _SD. At least 500 pollen grains were counted. **P<0.01 by Student's <i>t</i> test. (C) Recovery analysis of the pollen lethality phenotype. Ann5Pro-Ann5 fully rescued the sterile pollen phenotype in the Lat52-Ann5UTRi lines. Ann5Pro-G26 and Ann5Pro-G257 only partially rescued the aborted pollen genotype. Bright-field images were obtained with a microscope 1 h after germination. Red stars indicate the aborted pollen grains. Bars  = 20 µm.</p

<i>Ann5</i> overexpression did not influence pollen germination and tube growth in response to LatB.

<p>(A) Representative images of pollen grains and tubes from the Lat52-GFP, Lat52-Ann5-GFP 1 and 3 and Ann5Pro-Ann5 1 homozygous lines growing on germination medium containing 1 or 2 nM LatB for 3 h. Bar  = 50 µm. (B) Pollen germination ratios of the Lat52-GFP, Lat52-Ann5-GFP 1 and 3 and Ann5Pro-Ann5 1 homozygous lines after the pollen had germinated for 3 h in the presence of 1 or 2 nM LatB. Values represent the means ± _SD. Eight hundred pollen grains and tubes were counted. (C) Pollen tube lengths of the Lat52-GFP, Lat52-Ann5-GFP 1 and 3 and Ann5Pro-Ann5 1 homozygous lines after the pollen germinated normally for 2 h and then for 2 h in the presence of 2.5 or 5 nM LatB. Values represent the means ± _SD. Five hundred pollen tubes were measured.</p

Ann5 binds to negatively charged phospholipids and this association is stimulated by Ca²⁺.

<p>(A) Multiple sequence alignment of the deduced amino acid sequences of repeats I and IV from <i>Arabidopsis thaliana</i> AnnAt1 to 8. Ca²⁺-binding sites of type II GXGTD-(37 residues)-E/D are indicated by blue and red shadows. The residues with red asterisks indicate the locations of PCR-based site-directed mutagenesis. The mutants of repeats I and IV were named Ann5G26EG28E and Ann5G257EG259E, respectively, and the conserved glycine residues were replaced by glutamic acid residues. (B) Predicted three-dimensional structure of the Ann5 protein. The annexin core domain was composed of four homologous repeats that are colored in red (repeat I), green (repeat II), yellow (repeat III) and blue (repeat IV) and shaped as a slightly curved disc. The convex surface on which calcium ions bind (magenta spheres) participates in peripheral membrane binding. The type II Ca²⁺-binding sites are labeled with sticks, and the ribbon illustrates the highly α-helical structure. (C) Phospholipid-binding properties of the recombinant His6-Ann5, His6-Ann5G26EG28E, His6-Ann5G257EG259E and His6-Ann5G26EG28EG257EG259E proteins. The individual protein (50 µg) was incubated with liposomes (1∶1 PC/PS) in the presence of increasing Ca²⁺ concentrations. (-) denotes that the reaction mixtures contained neither liposomes nor Ca²⁺. (D) Comparison of the Ca²⁺-dependent phospholipid binding abilities of Ann5 and its mutants using densitometry analysis of the signal intensities of blots as described in (C). The phospholipid binding amount of Ann5 in 200 µM Ca²⁺ is normalized as 1 (control). The relative intensities are displayed as fold-binding over the control. Values represent mean ± _SD (n = 6).</p

Ann5 binds to actin filaments, as revealed by a high-speed co-sedimentation assay.

<p>(A) Either 0.6 µM (lanes 5 and 6) or 0.3 µM (lanes 7 and 8) Ann5 was incubated with 4 µM F-actin at 22°C for 1 h and then centrifuged at 100,000 g for 1 h. Equal amounts of the supernatant (S) and pellet (P) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue R. Samples in lanes 1 and 2 represent the supernatant and pellet of 0.6 µM Ann5 alone. Lanes 3 and 4 contain 4 µM F-actin alone. (B) The percentages of Ann5 in the supernatant and pellet were determined in the absence (lane 1 and 2) or presence (lane 5 and 6) of F-actin and are presented in (A) by densitometry. Values represent the means ± _SD (n = 9). **P<0.01 by Student's <i>t</i> test.</p

Quantitative analysis of pollen germination rates and pollen tube lengths of the Lat52-GFP, Lat52-Ann5-GFP 1 and 3, and Ann5Pro-Ann5 1 homozygous lines.

<p>For pollen germination rate, the pollen germinated for 3 h in the presence of 0.3 or 0.6 µM BFA. Eight hundred pollen grains and tubes were counted. For pollen tube length, the pollen germinated normally for 2 h and was then treated with 0.3 or 0.6 µM BFA for 2 h. A total of 500 pollen tubes were measured. Values represent the means ± _SD. *P<0.05 and **P<0.01 by Student's <i>t</i> test.</p

<i>Ann5G26EG28E</i> or <i>Ann5G257EG259E</i> overexpression decreased the resistance of pollen to BFA compared with <i>Ann5</i> overexpression.

<p>Pollen grains and tubes from the Lat52-GFP, Lat52-Ann5-GFP 1, Lat52-G26-GFP 1 and 2 and Lat52-G257-GFP 1 and 2 homozygous lines growing on germination medium containing 0.6 µM BFA in vitro. Medium containing 50 nM DMSO was used as the control. The images were captured after culturing for 3 h. Bar  = 50 µm.</p