CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA

Abstract

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.

Keywords: Arabidopsis; HDR; NHEJ; gene editing; knock-in.

Figure 1

The general scheme for obtaining lines with knock-in. Designations: nptII, neomycin phosphotransferase II gene providing plant cell resistance to kanamycin; dIFN, DNA sequence encoding the target dIFN protein; LF and RF, left and right flanking sequences homologous to the corresponding regions in the A. thaliana genome; sgRNA—single guide RNA gene; Cas9—endonuclease Cas9 gene; bar—phosphinothricin resistance gene; 12 KDa MSP—12 kDa subunit of microsomal signal peptidase (At4g40042) gene of A. thaliana; H3.3 histone—histone H3.3 gene HTR5 (At4g40040) of A. thaliana; NHEJ—non-homologous end joining; HDR—homologous directed repair.

Figure 2

Diagrams of genetic constructs for delivery of the dIFN gene to the region of the histone H3.3 gene. Designations: LF and RF, left and right flanking sequences homologous to the corresponding regions in the A. thaliana genome (intergenic region before the H3.3 histone gene); P1-P-NOS promoter of Agrobacterium tumefaciens nopaline synthase; P2, CaMV35S promoter of the cauliflower mosaic virus; nptII, neomycin phosphotransferase II gene providing plant cell resistance to kanamycin; S, is the DNA sequence encoding the leader signal of the carrot extensin gene, which ensures the transport of deltaferon into the apoplast; dIFN, DNA sequence encoding the target dIFN protein; GST, DNA sequence encoding a tag for protein affinity purification; sgRNA—Cas9 endonuclease recognition sites identical to the recognition site in the intergenic region upstream of the A. thaliana histone H3.3 gene, for excision of the construct from the plasmid in the cell.

Figure 3

Mutual arrangement of primers and restriction endonucleases used for the analysis of plant and plasmid DNA junctions. Designations: LB and RB, left and right borders of plant DNA; P1-P-NOS promoter of A. tumefaciens nopaline synthase; P2, CaMV35S promoter of the cauliflower mosaic virus; nptII, neomycin phosphotransferase II gene providing plant cell resistance to kanamycin; S is the DNA sequence encoding the leader signal of the carrot extensin gene, which ensures the transport of deltaferon into the apoplast; dIFN, DNA sequence encoding the target dIFN protein; Lo_plan3, Up_H3.3, Up_H3.3_1—primers used to identify the left border of the knock-in, the sizes of the resulting fragments are indicated above the arrows in bp; UpINS9178 (A), UpINS9368 (B), UpINS9525 (C), Lo10714 (D), Lo11187 (E), Lo11345 (F)—primers used to identify the right border of the knock-in by direct PCR, the sizes of the resulting fragments are indicated above the arrows in bp; MfeI, AsuII—restriction sites of enzymes used for the inverted PCR method, the size of restriction fragments is indicated in bp; BclI1, BclI2, BclI3, BclI4—primers used for inverted PCR.