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Mouse Dkk4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dkk4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dkk4 gene (NCBI Reference Sequence: NM_145592 ; Ensembl: ENSMUSG00000031535 ) is located on Mouse chromosome 8. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000033936). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dkk4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-127H3 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a conditional ready allele are phenotypically normal.

Exon 2 starts from about 16.89% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 953 bp, and the size of intron 3 for 3'-loxP site insertion: 824 bp. The size of effective cKO region: ~1217 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Dkk4 cKO region Exon of mouse Polb loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7717bp) | A(27.67% 2135) | C(22.25% 1717) | T(26.58% 2051) | G(23.51% 1814)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 + 22621998 22624997 3000 browser details YourSeq 180 25 1203 3000 94.6% chr6 + 107804966 107880486 75521 browser details YourSeq 164 611 1203 3000 85.7% chr11 + 116278695 116279100 406 browser details YourSeq 157 1031 1206 3000 96.6% chr2 + 156793981 156794167 187 browser details YourSeq 156 1036 1201 3000 97.6% chr2 + 25727648 25727823 176 browser details YourSeq 154 1031 1203 3000 94.8% chr5 + 33439651 33439847 197 browser details YourSeq 153 1030 1204 3000 94.3% chr2 + 100735483 100735692 210 browser details YourSeq 152 1027 1199 3000 95.4% chrX - 85059234 85059414 181 browser details YourSeq 152 1031 1199 3000 97.1% chr8 + 79855184 79855358 175 browser details YourSeq 152 1030 1299 3000 88.1% chr6 + 72531718 72531974 257 browser details YourSeq 148 705 1195 3000 86.4% chr9 - 40652030 40652434 405 browser details YourSeq 148 1031 1203 3000 93.5% chr17 - 60755510 60755682 173 browser details YourSeq 148 1026 1203 3000 90.4% chr5 + 113692252 113692421 170 browser details YourSeq 147 1031 1205 3000 93.1% chr5 - 118421461 118421684 224 browser details YourSeq 147 1063 1639 3000 84.5% chr15 + 34436771 34436934 164 browser details YourSeq 146 1031 1195 3000 95.2% chr8 - 23293975 23294141 167 browser details YourSeq 146 1032 1199 3000 94.1% chr10 + 65638192 65638365 174 browser details YourSeq 145 1032 1199 3000 93.4% chrX - 35858207 35858375 169 browser details YourSeq 145 1064 1232 3000 94.5% chr9 - 76089744 76089911 168 browser details YourSeq 145 1040 1204 3000 95.1% chr5 - 65657614 65657780 167

Note: The 3000 bp section upstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

BLAT Search Results (down)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 + 22626215 22629214 3000 browser details YourSeq 88 1560 1695 3000 90.1% chr16 + 37043736 37044056 321 browser details YourSeq 70 1587 1696 3000 90.7% chrX - 7636869 7636990 122 browser details YourSeq 69 1565 1665 3000 91.6% chr5 + 134325416 134325530 115 browser details YourSeq 65 1618 1695 3000 93.5% chr19 + 31634228 31634312 85 browser details YourSeq 64 1587 1670 3000 94.6% chr4 - 117686810 117686897 88 browser details YourSeq 64 1587 1672 3000 93.3% chr10 + 128754145 128754242 98 browser details YourSeq 64 1594 1696 3000 90.0% chr1 + 119506034 119506145 112 browser details YourSeq 64 1587 1663 3000 92.3% chr1 + 33989425 33989502 78 browser details YourSeq 63 1600 1717 3000 97.1% chr19 - 40515888 40516209 322 browser details YourSeq 63 1594 1672 3000 91.2% chr11 - 57822185 57822271 87 browser details YourSeq 63 1587 1665 3000 88.2% chr19 + 4728763 4728840 78 browser details YourSeq 62 1587 1663 3000 90.8% chr11 - 100309443 100309520 78 browser details YourSeq 62 891 970 3000 83.2% chr17 + 37020993 37021069 77 browser details YourSeq 61 1559 1661 3000 79.3% chr7 - 112879608 112879697 90 browser details YourSeq 61 1587 1687 3000 93.1% chr2 - 73177344 73177451 108 browser details YourSeq 61 1587 1664 3000 89.7% chr17 - 47160507 47160585 79 browser details YourSeq 61 1312 1658 3000 91.9% chr5 + 100504036 100504548 513 browser details YourSeq 61 1558 1651 3000 81.1% chr12 + 73683778 73683862 85 browser details YourSeq 60 1623 1705 3000 82.1% chr3 - 36420016 36420094 79

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Dkk4 dickkopf WNT signaling pathway inhibitor 4 [ Mus musculus (house mouse) ] Gene ID: 234130, updated on 24-Sep-2019

Gene summary

Official Symbol Dkk4 provided by MGI Official Full Name dickkopf WNT signaling pathway inhibitor 4 provided by MGI Primary source MGI:MGI:2385299 See related Ensembl:ENSMUSG00000031535 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Dkk-4 Expression Low expression observed in reference dataset See more Orthologs human all

Genomic context

Location: 8; 8 A2 See Dkk4 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (22623685..22628018)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (23734515..23738019)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 1 transcript

Gene: Dkk4 ENSMUSG00000031535

Description dickkopf WNT signaling pathway inhibitor 4 [Source:MGI Symbol;Acc:MGI:2385299] Location Chromosome 8: 22,624,043-22,627,547 forward strand. GRCm38:CM001001.2 About this gene This gene has 1 transcript (splice variant), 103 orthologues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dkk4-201 ENSMUST00000033936.7 1315 221aa ENSMUSP00000033936.6 Protein coding CCDS22180 Q8VEJ3 TSL:1 GENCODE basic APPRIS P1

23.50 kb Forward strand

22.615Mb 22.620Mb 22.625Mb 22.630Mb 22.635Mb Genes (Comprehensive set... Dkk4-201 >protein coding

Contigs < AC121835.2 Genes < Polb-201protein coding (Comprehensive set...

< Polb-202nonsense mediated decay

Regulatory Build

22.615Mb 22.620Mb 22.625Mb 22.630Mb 22.635Mb Reverse strand 23.50 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000033936

3.50 kb Forward strand

Dkk4-201 >protein coding

ENSMUSP00000033... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Pfam Dickkopf, N-terminal cysteine-rich Prokineticin domain

PANTHER Dickkopf-like protein

PTHR12113:SF10 Gene3D 2.10.80.10

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 200 221

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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