Fig. 1 PCR analysis of the S100a8 targeted locus. (A) Genomic structure of the targeted locus with positions of PCR primers (d1, d3, d4, d7, r3, r4, and r7). Intronic regions are represented as lines, and exons are represented as filled boxes numbered above. The oligonucleotide pairs Ad1 and Ar1 are not present in the mouse genome but introduced as diagnostic sequences together with the LoxP sites. The black bar below is the schematic representation of the donor DNA template; LoxP sites are represented as white boxes. Sizes of homology arms and PCR products obtained with different primer combinations on (B) to (E) are indicated. (B) PCR analysis of genomic DNA from F0 founder mice 1 to 20 (labeled above) using primer pair d3/r3 located outside of the DNA template homology arms (A). The PCR products of 715 and 543 bp correspond to the correctly targeted (founder 11 labeled by an arrow) and wild-type (animals 5, 8, 10, and 18) alleles of the S100a8 gene, respectively. The PCR products (>715 bp) presumably originating from multiple head-to-tail integrations of the DNA template were not detected. Size marker positions (in base pairs) are shown on the right. (C and D) PCR analyses of DNA samples from F0 founder mice 1 to 20 using primer pairs d3/Ar1 (C) and Ad1/r3 (D). (C) The PCR product of 257 bp corresponds to HDR integration of the 5′ homology arm detected in mouse samples 6 (labeled by arrow), 10, 11, 18, and 19. (D) The expected PCR product of 204 bp was detected in animals 6 (labeled by arrow), 7 to 9, and 11. In mouse numbers 10 and 18, the 3′ end of the DNA template integrated via NHEJ mechanism. (B to D) Genomic DNA from wild-type C57BL/6J mouse (wt) and water (neg) were used as controls. (E) PCR analysis at different annealing temperatures of genomic DNA from F0 founder number 6 using primer pair d4/r3. Only one PCR product of 750 bp, corresponding to a single copy targeted locus, was detected. A predicted PCR product for multiple head-to-tail DNA template amplification (~2247 bp) was not detected.
Fig. 2 Schematic representation of the S100a8 gene targeting strategy. (A) Wild-type mouse S100a8 locus. Exon 2 was chosen for elimination. Intronic and intergenic regions are represented as lines, and exons are represented as filled boxes numbered above. The vertical arrows indicate the target sites for the CRISPR-Cas9 complex with crRNA12 (C1) and crRNA3 (C2). The arrows marked with “B” correspond to Bam HI restriction endonuclease sites. The black bars below [marked “probe” in (A)] correspond to areas recognized by donor DNA–specific probes used in Southern blot analyses. The horizontal arrows denote the expected sizes of restriction DNA fragments given in kilobase. (B) Donor DNA template used in this study; the two LoxP sites are indicated by vertical arrows. (C) Genomic locus after HDR with single copy integration. (D) Targeted genomic locus with triple insertion of the donor DNA template. (E) Southern blot analysis of genomic DNA of the F1 offspring (36 to 45 and 47) hybridized with the template-specific probe [indicated in (A)]. Bam HI enzymatic digestion revealed the wild-type allele (4.0 kb) and three DNA fragments (3.2, 0.7, and 0.3 kb) corresponding to the targeted allele [marked in (C)]. DNA samples 36, 37, 39, 41, 43, 44, and 47 contain the correctly targeted S100a8 allele (S100a8 +/−). Samples 38, 40, 42, and 45 contain DNA fragments of 1.1 and 0.2 kb in size, indicating multiple copy head-to-tail integrations at the targeted locus [marked in (D)]. Size marker positions (in base pairs) are shown on the right. The DNA sample from the wild-type control mouse is indicated as “wt.”
Fig. 3 Different types and mechanisms of donor DNA integrations and multiplications. (A) Schematic representation of the loci for the cKO targeting strategy. Intronic regions are represented by gray lines, original exons are represented by filled boxes, exon X of donor DNA template is indicated as patterned boxes. Homology arms for HDR in the targeted locus are marked as black and red lines for left and right flanks, respectively. For better visualization, the respective homology arms of the donor DNA template are indicated by chess pattern. The target sites of the CRISPR-Cas9 complex are denoted as crRNA1 and crRNA2. (B) Integration of a single donor DNA template using HDR mechanism is shown. (C) Head-to-tail integration of donor DNA template is schematically drawn. (D and F) Different types of 5′ (D) and 3′ (F) integration events that were confirmed by sequencing are indicated. (E) Different types of observed repeat junctions of multiplied dsDNA and ssDNAs donor DNA templates are indicated. In addition, we analyzed the sequence of repeat junctions during head-to-tail multiplications of single stranded oligodeoxynucleotides (ODN, in Il4 locus, data not shown). Question mark (?) denotes a predicted but not experimentally verified scenario for dsDNA or ODN donors. (E a) We could not completely exclude that a rolling cycle mechanism for dsDNA or ssDNA multiplication could be responsible for at least part of the multimers where all ligated junctions are identical (cases from S100a8 and Treck1 loci). However, for a number of analyzed dsDNA-, ssDNA-, or ODN-derived head-to-tail repeats in the F1 generation, we identified various sequencing patterns within junction sites in each of the analyzed animals (E b-e). In a few cases, insertions of foreign DNA were observed at the junction sites between repeats (E f). Notably, head-to-head or tail-to-tail template multiplication was not observed. However, small inverted repeats originating from donor templates were observed within junction sites between head-to-tail repeats. This observation suggests that head-to-head or tail-to-tail ligations of DNA templates occur but are not stable in the locus and deleted (as inverted repeats) during cell division. In summary, obtained data indicate that NHEJ could be the major mechanism responsible for head-to-tail donor DNA multimerization.
Fig. 4 Analysis of F1 mice for LoxP site integration in the Il4 locus. (A) Schematic representation of the IL4 5′-LoxP DNA template. The genomic region is represented by lines, and the inserted artificial DNA sequence is indicated by an open rectangle. The 5′-LoxP site is designated by an arrow above, and the restriction endonuclease sites Bam HI (B) and Xho I (X) are indicated below. PCR primers are denoted by arrows. (B) PCR analysis of genomic DNA from selected F1 founder mice using the SD1/SR1 primer pair. (C) Schematic representation of a bidirectional primer strategy used to detect head-to-tail multiplication of donor DNA template. PCR primers are denoted by arrows, the repeat junction site is indicated by a black circle. (D) PCR analysis of genomic DNA from selected F1 founder mice using bidirectional primers SD1r and SR1d specifically detecting head-to-tail LoxP target DNA repeats.
- Table 1 Summary of cKO loci targeting and mechanisms of donor DNA integrations.
Gene name, the names for cKO-targeted genes are indicated [official ID provided by MGI (Mouse Genome Informatics)]; No. of F0 selected animals, number of F0 founders selected to contain a positively targeted allele; No. of F1 analyzed animals, number of analyzed mice from the F1 generation; No. of F1 positive SC animals, number of mice with correct HDR-HDR single copy donor template integration; No. of F1 positive MC animals, number of mice with identified multiple integrated copies of donor template; (F0), multiple copy integration of donor DNA template was identified in F0 founders; template size/strandedness (ss-ds DNA), donor DNA template sizes and strandedness are indicated; mechanism, mechanism for donor DNA template integration as determined; nd - not determined.
Gene name No. of F0 selected
animalsNo. of F1 analyzed
animalsNo. of F1 positive
SC animalsNo. of F1 positive
MC animalsTemplate size/
strandedness
(ss-ds DNA)Mechanism S100a8 2 14 (No.6)
7 (No.11)9 (No.6)
4 (No.11)5
3ssDNA
(PCR)
591 ntHDR-HDR
NHEJ-HDR
NHEJ-NHEJTrek1 2 21 0 16 dsDNA
1257 bpHDR-NHEJ
NHEJ-HDRTrek1 6 28 0 12 ssDNA (PCR), 1257 nt HDR-HDR, NHEJ-HDR, HDR-NHEJ Trek1 3 26 0 3(F0) ssDNA
(IDT)
1286 ntnd Inf2 11 34 2 15 ssDNA
(PCR)
711 ntHDR-HDR Trpc6 3 22 5 nd dsDNA
880 bpHDR-HDR Trpc6 4 34 5 2 ssDNA
(PCR)
880 ntHDR-HDR Ccnd2 1 46 19 1(F0) dsDNA
1658 bpHDR-HDR Il4_5′LoxP 18 49 19 30 ssDNA
(PCR)
210 bpHDR-HDR Il4_flox 4 41 0 1(F0) ssDNA
(PCR)
1258 ntNHEJ-HDR Total F1: 63 (~43%) Total F1: 83 (~57%)
Supplementary Materials
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/7/eaax2941/DC1
Supplementary Material and Methods
Fig. S1. Evaluation of in vivo S100a8 crRNA cleaving efficiency in mouse embryos.
Fig. S2. PCR analysis of genomic DNA from F0 founder number 6 after HTTP integration in the S100a8 locus at different touch down/annealing temperature conditions using primer pair (d4/r4) (Fig. 1D).
Fig. S3. Sequence analysis of heterozygous animal (F1) number 45 with MC head to tail integration of the DNA template in the S100a8 gene (Figs. 1E and 2A).
Fig. S4. Analysis of the Inf2 targeted locus.
Fig. S5. Analysis of the Trek1 targeted locus.
Fig. S6. Analysis of the Trpc6 targeted locus.
Fig. S7. Analysis of the Ccnd2 targeted locus.
Table S1. List of crRNAs used.
Table S2. Designed donor DNA templates.
Table S3. List of oligonucleotides used for ssDNA donor template generation by asymmetric PCR and PCR analyses of targeted loci.
Additional Files
Supplementary Materials
This PDF file includes:
- Supplementary Material and Methods
- Fig. S1. Evaluation of in vivo S100a8 crRNA cleaving efficiency in mouse embryos.
- Fig. S2. PCR analysis of genomic DNA from F0 founder number 6 after HTTP integration in the S100a8 locus at different touch down/annealing temperature conditions using primer pair (d4/r4) (Fig. 1D).
- Fig. S3. Sequence analysis of heterozygous animal (F1) number 45 with MC head to tail integration of the DNA template in the S100a8 gene (Figs. 1E and 2A).
- Fig. S4. Analysis of the Inf2 targeted locus.
- Fig. S5. Analysis of the Trek1 targeted locus.
- Fig. S6. Analysis of the Trpc6 targeted locus.
- Fig. S7. Analysis of the Ccnd2 targeted locus.
- Table S1. List of crRNAs used.
- Table S2. Designed donor DNA templates.
- Table S3. List of oligonucleotides used for ssDNA donor template generation by asymmetric PCR and PCR analyses of targeted loci.
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