The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli

Abstract

Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free.

Figure 1. General outline of the no-SCAR method.

On day 1 the pCas9cr4 plasmid is used to transform E. coli, followed by plating on LB + Cm, and growth at 37 °C. On day 2 the resulting strain can be transformed with pKDsg-xxx plasmid, where –xxx denotes the targeted gene, plated on LB + Spec and Cm, and incubated at 30 °C overnight. On day 3 the resulting strain is grown in SOB until OD ~0.5 and λ-red is induced with 50 mM L-arabinose. After 15–20 minutes the cells are made electrocompetent and transformed with ssDNA or dsDNA that confers a mutation to the protospacer or PAM sequence. After 1–2 hours of recovery the cells are plated on LB + Spec, Cm, and aTc, then incubated at 30 °C overnight. On day 4 colonies are screened by PCR and grown at 37 °C to cure the pKDsg-xxx plasmid. The next pKDsg-xxx plasmid is then used to transform the mutant strain on Day 5 and the process is repeated.

Figure 2. Schematic map of the no-SCAR plasmids.

(A) Schematic of the plasmid pCas9cr4 which has cas9 expressed under control of the P_TET promoter and tetR constitutively expressed. (B) Schematic of the plasmid pKDsg-xxx which has the sgRNA expressed under control of the P_TET promoter and the three genes that compose the λ-Red system under control of the arabinose inducible promoter P_araB.

Figure 3. Schematic of the genomic DNA modifications demonstrated by the no-SCAR system.

The donor and target DNA are on the left, the chimeric genomic DNA produced after integration through a fully single-stranded DNA intermediate is in the middle, and the segregated chimeric DNA that results in populations of mutant and wild-type are on the right. (a,b) 21 bp of the targeted region for making point mutations into the rpoB and ack genes. The PAM site is shown in red, mutations are shown in lowercased blue text, and mutation target in lowercased black text. (c) Deletion of 1095 bp of ack using a 73mer oligo. (d) Insertion of 79 bp at the C-terminus of pfkA using dsDNA. Red × indicates the site of Cas9 targeting. (e) Insertion of degeneracies outside of the protospacer sequence.