Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases

Abstract

Programmable CRISPR-Cas systems have augmented our ability to produce precise genome manipulations. Here we demonstrate and characterize the ability of CRISPR-Cas derived nickases to direct targeted recombination of both small and large genomic regions flanked by repetitive elements in Escherichia coli. While CRISPR directed double-stranded DNA breaks are highly lethal in many bacteria, we show that CRISPR-guided nickase systems can be programmed to make precise, nonlethal, single-stranded incisions in targeted genomic regions. This induces recombination events and leads to targeted deletion. We demonstrate that dual-targeted nicking enables deletion of 36 and 97 Kb of the genome. Furthermore, multiplex targeting enables deletion of 133 Kb, accounting for approximately 3% of the entire E. coli genome. This technology provides a framework for methods to manipulate bacterial genomes using CRISPR-nickase systems. We envision this system working synergistically with preexisting bacterial genome engineering methods.

Keywords: CRISPR; Cas9 nickase; chromosome deletion; direct repeats; genome engineering; recombination.

Figure 1

CRISPR-directed SSBs are not lethal to K12 E. coli. (a) A schematic representation of sgRNA guided Cas9s targeting a chromosomally integrated GFP reporter. The sgRNA guide sequence is visualized adjacent to its target sequence. The protospacer adjacent motif (PAM) sequence is highlighted in red. Arrows denote where DNA cleavage occurs by two Cas9 mutants, respectively. Cas9^D10A cleaves the target DNA strand of the sgRNA and Cas9^H840A cleaves the nontarget strand. (b) Transformation efficiencies of sg(C1) (gray) or untargeted sg(−) (white) in K12 E. coli containing Cas9^WT, Cas^D10A, Cas9^H840A or dCas9. Error bars represent ± standard deviation (n = 3).

Figure 2

Nick-induced recombination leads to single gene chromosomal deletion. (a) A diagram of chromosomally integrated dual-fluorescent reporter with internal homologous DNA sequences (R), shown as dark and light gray boxes. sgRNA-guided Cas9 nicking enzyme generates an SSB, illustrated by the scissor. CRISPR induced deletions of GFP results in the joining of two repeats, shown as a gray box with both dark and light shades. Primers (P^r1 and P^r4) flank the dual fluorescence reporter. (b) Flow cytometry screening of pooled sgRNA transformations for homologous recombination. Histograms show population fluorescence distributions for controls sg(−) (green), cells containing RFP with no GFP (gray), and sg(T1) coexpressed with Cas9^D10A (red). Vertical dashed line represents threshold for GFP expression. 86% of sg(T1) cells fall to the left of the line, indicating the loss of GFP expression. (c) Percent of flow cytometry estimated GFP deletion for various sgRNAs targeting the noncoding strand using Cas9^D10A (blue) and Cas9^H840A (orange). Nicking sites within GFP are represented by blue and orange triangles and black squares representing sgRNA target sites. Cut positions are defined as bp from the left repeat (d) Fluorescence microscopy interrogation of dual-fluorescent reporter expression for sg(−) and sg(T1) transformants in E. coli expressing Cas9^D10A. GFP expression is undetectable in sg(T1) while RFP expression is similar in both sg(−) and sg(T1). Phase contrast images show cells have normal morphology and RFP expression. (e) Gel-electrophoresis of amplicons using primers P^r1 and P^r4 from transformants for sg(T1) and sg(−). The primers generate a 4.6 kilobase (Kb) amplicon for the initial RFP:GFP site, and following HR the primers generate a 3.6 Kb amplicon. Six out 6 sg(T1) transformants amplify at sizes of approximately 3.6 kilobases, while a sg(−) control template results in banding at 4.6 Kb.

Figure 3

Nickase directed deletion of a 36 kilobase genomic region. (a) Top half is a schematic representation of a 36.8 Kb genomic region with two IS5 direct-repeats (gray, R) on the K12 MG1655 chromosome. Colored boxes serve as visual aids to mark relative positions and sizes of various genomic regions. A series of 10 sgRNAs were designed to target various locations (black squares, sg(A–J)). The position of black squares indicate which strand is targeted by the sgRNA with I, A and H targeting the + strand, and J and B-G targeting the -. The distances between sgRNA targets are shown in panel C. Primers flanking each repeat (P^h1–P^h4) are used for PCR based genotyping of chromosomal deletions. The His-biosynthetic operon (His-Operon) (orange) located between the IS5 elements serves as a screenable marker for cells harboring deletions. Bottom half represents results of CRISPR induced deletion of genomic region between two repeats (shown gray box with mixed shades). (b) A schematic representation of multitargeting nicking systems around the left IS5, with 3′ Overhang (OH) and 5′ OH cut orientations highlighted. Bp apart is the base pair distance between cut sites. Blue triangles indicate cut sites for respective guides. (c) PCR screening and gel electrophoresis of pooled transformations to detect formation of recombined site (red, gray yellow icon) using primers P^h1 and P^h4 (d) Histidine auxotrophy screening of individual isolated transformants expressing Cas9^D10A with sg(B:I) and sg(A:B). Differential growth on M9 minimal medium containing synthetic complete amino acids (M9SC) or M9 without histidine (M9-H) suggests deletions of the targeted region when no colonies are formed in M9-H. Colony positions on M9SC correspond to those on M9-H. Success rates are denoted as the number of colonies unable to grow in M9-H over the total number of colonies (e) PCR genotyping of 6 sg(B:I) histidine auxtrophs and a control sg(−) transformant using 3 different primer combinations confirming formation of the recombined site (bottom row).

Figure 4

Multiplex targeted genome remodeling achieves 133 Kb deletion. (a) A schematic view of two separate genomic regions surrounded by direct repeat sequences. Definitions of illustrations are the same as in Figure 3. The middle disjoint beige box represents 670 Kb distance between these two regions. The left region is 97 kilobases and contains the tryptophan biosynthesis operon (TRP) and the pyrF gene (labeled bright blue boxes). Recombination between these repeats results in deletion of 97 Kb (Δ97 Kb) and tryptophan auxotrophy. The remodeled site (purple, gray and green) brings primers P^t1 and P^t4 in into close proximity. sgRNAs K and L target the sequences directly adjacent to the left repeat to this region. In parallel, guides B and I target a separate region containing His (orange box). The position of black squares indicate which strand is targeted by the sgRNA with K and I targeting the + strand and L and B targeting the – strand (see Figure 3 also). Deletion of this region results in a loss of 36 Kb (Δ36 Kb) and histidine auxotrophy. The remodeled sequence (red, gray and yellow) results in close proximity of P^h1 and P^h4. (b) PCR monitoring of HR in Cas9^D10A expressing cells. Primers P^t1 and P^t4 detect deletion of 97 Kb (purple, gray and green icon, resulting in a 1.5 Kb amplicon). P^h1 and P^h4 detect recombination across 36 Kb (red, gray and yellow icon, resulting in a 1.8 Kb amplicon). Different sgRNA targets are indicated beneath the gel photo. sg(−) is a guide sequence not matching the bacterial genome. Amplification indicates occurrence of recombination. (c) Resulting phenotypes of isolated E. coli containing different remodeling combinations. Cells isolated containing different deletions replica plated on M9 synthetic complete medium (M9SC), M9 lacking histidine (M9-H), and M9 without tryptophan (M9-T). Vertical columns correspond to different deletions. sg(−) with no genomic deletion (Δ0) serves as the control. sg(B:I:K:L) expressing cells can harbor 36 (Δ36), 97 (Δ97) or combined 133 kilobase (Δ133) deletions. Δ36, Δ97 and Δ133 are auxotrophic histidine, tryptophan and both, respectively.