Consequences of Cas9 cleavage in the chromosome of Escherichia coli

Abstract

The RNA-guided Cas9 nuclease from CRISPR-Cas systems has emerged as a powerful biotechnological tool. The specificity of Cas9 can be reprogrammed to cleave desired sequences in a cell's chromosome simply by changing the sequence of a small guide RNA. Unlike in most eukaryotes, Cas9 cleavage in the chromosome of bacteria has been reported to kill the cell. However, the mechanism of cell death remains to be investigated. Bacteria mainly rely on homologous recombination (HR) with sister chromosomes to repair double strand breaks. Here, we show that the simultaneous cleavage of all copies of the Escherichia coli chromosome at the same position cannot be repaired, leading to cell death. However, inefficient cleavage can be tolerated through continuous repair by the HR pathway. In order to kill cells reliably, HR can be blocked using the Mu phage Gam protein. Finally, the introduction of the non-homologous end joining (NHEJ) pathway from Mycobacterium tuberculosis was not able to rescue the cells from Cas9-mediated killing, but did introduce small deletions at a low frequency. This work provides a better understanding of the consequences of Cas9 cleavage in bacterial chromosomes which will be instrumental in the development of future CRISPR tools.

Figure 1.

Weak self-targeting CRISPR-Cas9 systems can be tolerated through homology directed repair. (A) Position of the targets on the Escherichia coli chromosome. Targets on the inside of the circle are on the non-template strand of the gene, targets on the outside are on the template strand. (B) The pCRRNA carrying different spacers was transformed in cells expressing Cas9 constitutively. Average CFU numbers are reported for transformation in wild-type cells (black bars) and recA-cells (gray bars), showing that some spacers can be tolerated in the presence of recA but not in the recA-strain (mean ± s.d., n ≥ 3). Transformation events yielding small colonies are marked with a hashtag (see Supplementary Figure S1 for colony size measurments). (C) Schematics of the transformation assay performed to demonstrate homology directed repair. The pCas9 plasmid carrying Cas9, the tracrRNA and a CRISPR array was programmed to target a position within the lacZ gene. The resulting plasmid pCas9::lacZ2 was transformed in cells carrying a plasmid with homologies to the target region but carrying a mutation preventing Cas9 cleavage (pLCX). (D) CFU numbers are reported after transformation either in wild-type (black bars) or recA-cells (gray bars), showing that the presence of a repair template rescues cells targeted by Cas9 at the lacZ2 position (mean ± s.d., n ≥ 3).

Figure 2.

Cas9 cleavage in the chromosome induces the SOS response. (A) Schematics of the SOS reporter assay. Cas9 is expressed under the leaky control of a non-induced ptet promoter in the chromosome. The tracrRNA and crRNA are expressed from the pCRRNA plasmid. Cas9-mediated breaks lead to the formation of recA/ssDNA filaments which activate the self-cleavage of the LexA repressor. This releases the repression of GFPmut2 controlled by the sulA promoter. (B) The pCRRNA plasmid programmed to target the lacZ1 position (black bars) or a control empty pCRRNA (gray bars) were introduced in different cell backgrounds. GFP fluorescence was measured during exponential growth (mean ± s.d., n ≥ 3). (C) SOS response resulting from CRISPR targeting with different spacers. The bar marked as ‘control’ indicates the auto-fluorescence level of Escherichia coli without the pZA31-sulA-GFP plasmid. Spacers that cannot be transformed under constitutive Cas9 expression from the pCas9 (see Figure 1B) are shown in white. Spacers that can be transformed but lead to the formation of small colonies (see Figure 1B) are shown in gray. Finally, spacers that can be transformed in the presence of pCas9 and form colonies of regular size (see Figure 1B) are shown in black (mean ± s.d., n ≥ 3).

Figure 3.

Mu Gam enhances Cas9-mediated killing. (A) Schematics showing the outcome of weak or strong Cas9 cleavage activity in the chromosome of Escherichia coli. Weak targeting leaves a chromosomal copy intact that can be used as a template to repair the broken chromosome through HR. The repaired chromosome can then be cut again, thus entering a cycle of cleavage and repair, which constitutively induces the SOS response. Strong targeting leads to the simultaneous cleavage of all copies of chromosome which cannot be repaired through HR and thus leads to cells death. The Mu Gam protein can specifically bind to double stranded ends and block repair through HR, thereby promoting cell death even under weak targeting conditions. (B) Plasmid pCRRNA carrying the lacZ1, lacZ2 spacers or a spacer-less control was transformed in cells carrying plasmid pLC13 expressing Mu Gam under the control of an arabinose inducible promoter. The number of transformants obtained in the presence or absence of arabinose is reported (mean ± s.d., n ≥ 3). Upon Gam expression both weak and strong targeting leads to efficient killing.

Figure 4.

Cas9 cleavage of the lacZ gene induces the formation of large deletions. (A) The pCRRNA plasmid carrying the lacZ1, lacZ2 spacers or a spacer-less control was transformed in cells expressing Cas9 constitutively from the pCas9 plasmid. The number of white and blue CFU on Xgal are reported (white bars and grey bars), indicating the number of colonies where lacZ was inactivated (mean ± s.d., n ≥ 3). (B) Schematics of the lac operon genomic region. The target position is shown by a black arrow. The deletions mapped in 14 white colonies are depicted by black bars.

Figure 5.

NHEJ repair of Cas9 induced DSB in the genome of Escherichia coli. (A) Plasmid pCas9 programmed with spacer lacZ2 or a spacer-less control was transformed in recB-cells with or without the NHEJ system (Ku and LigD) from Mycobacterium tuberculosis. The number of blue and white CFUs on Xgal plates is reported (grey and white bars), indicating colonies where lacZ was inactivated. (B) Schematics showing the beginning of the lacZ open-reading frame. Deletions mapped in one blue and 13 white colonies are depicted by black bars. Base numbers from the start codon are shown. (C) The right edge of the mapped deletions is aligned below the crRNA and target DNA for spacer lacZ2. The cleavage point is shown by a vertical black bar and bold characters indicate the PAM motif.