CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations

Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended mutations are frequently found over 200 kb from the intended edit site and even over 10 kb from potential off-target sites. We attribute the high frequency of unintended mutations to error-prone polymerases expressed in response to dsDNA breaks introduced at the edit site. Most unintended mutations occur in regulatory or coding regions and thus may have phenotypic effects. Our findings highlight the risks associated with genome editing techniques involving dsDNA breaks in E. coli and likely other bacteria and emphasize the importance of sequencing the genomes of edited cells to ensure the absence of unintended mutations.


Supplemental Figures
red, lambda Red proteins Exo, Beta and Gam under regulation of a heat-inducible promoter.pSC101 temperature-sensitive origin of replication allows plasmid to be maintained at 30 °C and cured at 37 °C.Constructed from pSS9_gRNA (1).Purple, promoter for sgRNA; cyan, 20-nucleotide spacer sequence targeting sequences; red, sgRNA scaffold; dark and light green, primer pairs used to amplify the backbone for guide and donor plasmids, respectively.Insertion site for editing cassettes used in donor plasmids is immediately upstream of the promoter.(yellow) and a short downstream HA (small cyan HA2); second, a segment containing two synthetic terminators (the synthetic terminators ensure efficient cleavage at the I-SceI site), an I-SceI cut-site (green) and a streptomycin resistance gene (grey); and third, a segment containing a short HA upstream of the edit (small red HA1), the edit (yellow) and a downstream HA (large blue HA2).Dark green, primers used to amplify the editing cassette.

Figure S2 .
Figure S2.pAM041, the plasmid from which backbones for donor and guide plasmids were amplified.

Figure S3 .
Figure S3.pKAW086, which contains the editing cassette used in lambda Red recombineering experiments.Red and cyan, regions with homology upstream and downstream of E. coli pdxB, respectively; dark green, primers used to amplify the editing cassette.

Figure S4 .
Figure S4.pKAW066, which contains the editing cassette used in I-SceI and is comprised of three segments: first, a segment containing a homology arm (HA) upstream of the edit (large red HA1), the edit

Figure S5 .
Figure S5.pKAW109, the plasmid used in control experiments.The backbone was amplified from pAM041 (Figure S2) but lacks an editing cassette and sgRNA.

Table S1 .
Strains used in this work.All strains except DH5a and DH5a lambda pir contained the helper plasmid pDY118A.Intm designates intermediate strains that had previously been edited and were used for a second round of genome editing.

Table S2 .
Plasmids used in this work.
pDY348sgRNA targets an mCherry-AGG sequence introduced at codon 499 of cyaA through a previous round of genome editing; repair template introduces two synonymous mutations at codons 497 and 498 (Table S4, GB_cyaA499_2nd) this study pDY449 sgRNA targets a sequence of synonymous codons introduced at codons 84-92 of pykF by a previous round of genome editing; repair template introduces two synonymous mutations at codons 85 and 86 (Table S4, GB_pykF085_2nd) (3) pDY450 sgRNA targets a sequence of synonymous codons introduced at codons 84-92 of pykF by a previous round of genome editing; repair template introduces five synonymous mutations at codons 83, 84 and 90 (Table S4, GB_pykF090_2nd) (3) pDY452 sgRNA targets a sequence of synonymous codons introduced at codons 453-459 of pykF by a previous round of genome editing; repair template introduces seven synonymous mutations at codons 452, 453 and 466 (Table S4, GB_pykF466_2nd) (3) pDY453 sgRNA targets a sequence of synonymous codons introduced at codons 453-459 of pykF by a previous round of genome editing; repair template introduces four synonymous mutations at codons 452 and 453 (Table S4, GB_pykF470_2nd)

Table S3 .
PCR primers used in this work.F and R designate forward and reverse primers, respectively.

Table S4 .
gBlocks used to construct donor plasmids used for Cas9-assisted editing.Lower-case italics, extensions to direct Gibson Assembly with vector backbone; upper-case black, editing cassette; green, J23119 constitutive promoter; blue, 20-nt gRNA spacer sequence; red and magenta, target and synonymous immunizing mutations in or near the PAM site, respectively.(Target codons are underlined.)

Table S5 .
120-nt ssDNA editing cassettes used for Cas9-assisted editing and the associated spacer sequences used to direct Cas9 genome cleavage.Red and magenta, target and immunizing mutations, respectively.Target codons are underlined.

Table S7 .
Editing cassette used to insert T at position 98 in rpoS with I-SceI-assisted editing Red, homology arm (HA) upstream of the edit; yellow, edit; cyan, downstream HA; black, segment containing terminators; green, segment containing an I-SceI cut-site; black, segment containing a promoter; grey, streptomycin resistance gene; red, short HA upstream of the edit; yellow, edit; blue,

Table S8 .
60-nt oligonucleotides containing spacer sequences used to make guide plasmids Uppercase, 20-nt spacer; lowercase, sequences that overlap the guide plasmid backbone.The complementary 60-nt oligonucleotide fragments were annealed and then ligated into the pAM041 backbone by Gibson assembly.