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Mouse Odf2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Odf2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Odf2 gene (NCBI Reference Sequence: NM_001177659 ; Ensembl: ENSMUSG00000026790 ) is located on Mouse chromosome 2. 21 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 21 (Transcript: ENSMUST00000113759). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Odf2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-252F14 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 gene trapped allele exhibit embryonic lethality before implantation and transmission ratio distortion while all heterozygous males display normal development and fertility. Males heterozygous for other alleles are either infertile orshow reduced fertility.

Exon 5 starts from about 9.48% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 7507 bp, and the size of intron 6 for 3'-loxP site insertion: 979 bp. The size of effective cKO region: ~1765 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 5 6 7 8 21 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Odf2 Homology arm cKO region 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(8265bp) | A(23.96% 1980) | C(22.87% 1890) | T(27.77% 2295) | G(25.41% 2100)

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% chr2 + 29897828 29900827 3000 browser details YourSeq 140 1786 2265 3000 82.9% chr2 - 29961767 29962140 374 browser details YourSeq 139 99 693 3000 91.6% chr18 - 65511564 65899363 387800 browser details YourSeq 138 1779 2257 3000 90.2% chr4 - 135988760 135989391 632 browser details YourSeq 135 1734 1918 3000 91.0% chr11 + 119423527 119423757 231 browser details YourSeq 133 1724 1914 3000 85.3% chr13 - 54141983 54142161 179 browser details YourSeq 132 1734 1914 3000 92.9% chr6 + 5556452 5557031 580 browser details YourSeq 125 1726 1889 3000 92.8% chr14 - 121629373 121629534 162 browser details YourSeq 125 1724 1889 3000 92.0% chr3 + 126593945 126594106 162 browser details YourSeq 120 1729 1889 3000 92.7% chrX - 106070000 106070159 160 browser details YourSeq 120 1726 1889 3000 90.5% chr6 - 108179758 108179916 159 browser details YourSeq 120 1 202 3000 91.2% chr6 - 85528727 85529073 347 browser details YourSeq 119 2 194 3000 90.5% chr14 - 52156696 52156906 211 browser details YourSeq 117 1 194 3000 90.4% chr17 - 35769928 35770257 330 browser details YourSeq 117 1779 1919 3000 91.5% chr15 - 53991174 53991314 141 browser details YourSeq 116 1 194 3000 91.4% chr1 + 194191440 194191741 302 browser details YourSeq 115 1726 1914 3000 90.2% chr3 - 66085676 66085888 213 browser details YourSeq 114 1 193 3000 92.0% chr2 + 166990083 166990345 263 browser details YourSeq 113 1 197 3000 90.0% chrX + 60222617 60223019 403 browser details YourSeq 108 1794 1919 3000 92.9% chr3 + 129771518 129771643 126

Note: The 3000 bp section upstream of Exon 5 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% chr2 + 29902593 29905592 3000 browser details YourSeq 40 2253 2536 3000 71.5% chr2 + 106979993 106980258 266 browser details YourSeq 38 1689 1734 3000 95.5% chr14 - 56270671 56270720 50 browser details YourSeq 34 2928 2963 3000 97.3% chr16 - 18657075 18657110 36 browser details YourSeq 34 1691 1730 3000 97.3% chr11 + 63308768 63308815 48 browser details YourSeq 33 1750 1838 3000 92.4% chr16 - 21974746 21974834 89 browser details YourSeq 32 1691 1734 3000 73.0% chr5 - 103328890 103328926 37 browser details YourSeq 30 1694 1734 3000 77.8% chr5 - 100185601 100185637 37 browser details YourSeq 27 2500 2533 3000 91.2% chr5 + 122181579 122181613 35 browser details YourSeq 26 2524 2553 3000 93.4% chr2 - 37447638 37447667 30 browser details YourSeq 26 1696 1731 3000 86.2% chr14 - 13332583 13332618 36 browser details YourSeq 26 2231 2274 3000 86.7% chr11 + 51891971 51892013 43 browser details YourSeq 24 700 724 3000 100.0% chr12 - 20237900 20237926 27 browser details YourSeq 23 1649 1673 3000 87.5% chr6 - 33248241 33248264 24 browser details YourSeq 20 1371 1390 3000 100.0% chr11 + 95211466 95211485 20

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Odf2 outer dense fiber of sperm tails 2 [ Mus musculus (house mouse) ] Gene ID: 18286, updated on 24-Oct-2019

Gene summary

Official Symbol Odf2 provided by MGI Official Full Name outer dense fiber of sperm tails 2 provided by MGI Primary source MGI:MGI:1098824 See related Ensembl:ENSMUSG00000026790 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as AI848335; MMTEST29 Expression Biased expression in testis adult (RPKM 224.3), cerebellum adult (RPKM 22.9) and 4 other tissues See more Orthologs human all

Genomic context

Location: 2 B; 2 20.79 cM See Odf2 in Genome Data Viewer

Exon count: 25

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (29889014..29931746)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (29745240..29787266)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 25 transcripts

Gene: Odf2 ENSMUSG00000026790

Description outer dense fiber of sperm tails 2 [Source:MGI Symbol;Acc:MGI:1098824] Gene Synonyms MMTEST29, cenexin Location Chromosome 2: 29,889,221-29,931,746 forward strand. GRCm38:CM000995.2 About this gene This gene has 25 transcripts (splice variants), 229 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Odf2- ENSMUST00000046571.13 3959 825aa ENSMUSP00000049272.7 Protein coding CCDS50556 A3KGV1 TSL:2 202 GENCODE basic APPRIS ALT1

Odf2- ENSMUST00000113756.7 3826 825aa ENSMUSP00000109385.1 Protein coding CCDS50556 A3KGV1 TSL:1 204 GENCODE basic APPRIS ALT1

Odf2- ENSMUST00000113759.8 3807 826aa ENSMUSP00000109388.2 Protein coding CCDS50555 A3KGV1 TSL:1 206 GENCODE basic APPRIS ALT1

Odf2- ENSMUST00000113764.3 2450 638aa ENSMUSP00000109393.3 Protein coding CCDS15861 A3KGV1 TSL:5 209 GENCODE basic APPRIS P3

Odf2- ENSMUST00000028128.12 2376 638aa ENSMUSP00000028128.6 Protein coding CCDS15861 A3KGV1 TSL:1 201 GENCODE basic APPRIS P3

Odf2- ENSMUST00000113763.7 2364 638aa ENSMUSP00000109392.1 Protein coding CCDS15861 A3KGV1 TSL:5 208 GENCODE basic APPRIS P3

Odf2- ENSMUST00000113755.7 2305 652aa ENSMUSP00000109384.1 Protein coding CCDS50557 A3KGV1 TSL:1 203 GENCODE basic

Odf2- ENSMUST00000113767.7 2249 701aa ENSMUSP00000109396.1 Protein coding CCDS50554 A3KGW0 TSL:1 211 GENCODE basic APPRIS ALT1

Odf2- ENSMUST00000113765.7 3188 830aa ENSMUSP00000109394.1 Protein coding - A3KGV1 TSL:5 210 GENCODE basic APPRIS ALT1

Odf2- ENSMUST00000113757.7 3082 806aa ENSMUSP00000109386.1 Protein coding - A3KGV1 TSL:5 205 GENCODE basic

Odf2- ENSMUST00000113762.7 2353 657aa ENSMUSP00000109391.1 Protein coding - A3KGV9 TSL:5 207 GENCODE basic

Odf2- ENSMUST00000137558.7 814 271aa ENSMUSP00000117887.1 Protein coding - F6Y325 CDS 5' and 3' 218 incomplete TSL:3

Odf2- ENSMUST00000133233.7 658 138aa ENSMUSP00000117628.1 Protein coding - A3KGV3 CDS 3' incomplete 217 TSL:2

Odf2- ENSMUST00000123335.7 403 69aa ENSMUSP00000121207.1 Protein coding - A3KGV2 CDS 3' incomplete 212 TSL:3

Odf2- ENSMUST00000184845.7 3035 680aa ENSMUSP00000139390.1 Nonsense mediated - V9GXZ0 TSL:5 225 decay

Odf2- ENSMUST00000152503.1 4291 No - Retained intron - - TSL:1 222 protein

Odf2- ENSMUST00000153216.7 828 No - Retained intron - - TSL:2 224 protein

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Odf2- ENSMUST00000152026.1 673 No - Retained intron - - TSL:3 221 protein

Odf2- ENSMUST00000131165.7 553 No - Retained intron - - TSL:5 216 protein

Odf2- ENSMUST00000148883.1 437 No - Retained intron - - TSL:2 219 protein

Odf2- ENSMUST00000152932.7 793 No - lncRNA - - TSL:5 223 protein

Odf2- ENSMUST00000129960.7 674 No - lncRNA - - TSL:3 214 protein

Odf2- ENSMUST00000126103.7 589 No - lncRNA - - TSL:2 213 protein

Odf2- ENSMUST00000130899.1 395 No - lncRNA - - TSL:3 215 protein

Odf2- ENSMUST00000150827.1 349 No - lncRNA - - TSL:3 220 protein

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62.53 kb Forward strand 29.88Mb 29.90Mb 29.92Mb 29.94Mb Genes (Comprehensive set... Cercam-201 >protein coding Odf2-212 >protein coding Odf2-214 >lncRNA Odf2-221 >retained intron

Cercam-205 >retained intron Odf2-219 >retained intron Odf2-223 >lncRNA Gle1-207 >retained intron

Odf2-211 >protein coding Gle1-205 >retained intron

Odf2-208 >protein coding Gle1-201 >protein coding

Odf2-204 >protein coding

Odf2-205 >protein coding

Odf2-225 >nonsense mediated decay

Odf2-213 >lncRNA Odf2-216 >retained intron

Odf2-217 >protein coding Odf2-222 >retained intron

Odf2-206 >protein coding

Odf2-203 >protein coding

Odf2-224 >retained intron

Odf2-218 >protein coding

Odf2-202 >protein coding

Odf2-201 >protein coding

Odf2-220 >lncRNA

Odf2-207 >protein coding

Odf2-210 >protein coding

Odf2-209 >protein coding

Odf2-215 >lncRNA

Contigs AL928926.9 > Genes < Gm23865-201snRNA (Comprehensive set...

Regulatory Build

29.88Mb 29.90Mb 29.92Mb 29.94Mb Reverse strand 62.53 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000113759

41.93 kb Forward strand

Odf2-206 >protein coding

ENSMUSP00000109... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF90257

PANTHER Outer dense fibre protein 2-related

PTHR23162:SF8

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

Variant Legend

missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 826

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

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