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Mouse Osr2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Osr2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Osr2 gene (NCBI Reference Sequence: NM_054049 ; Ensembl: ENSMUSG00000022330 ) is located on Mouse chromosome 15. 4 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000022952). 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 Osr2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-267M8 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: Homozygous null mice display neonatal lethality, cleft of the secondary palate, and thickened tympanic rings.

Exon 2~3 is not frameshift exon, and covers 91.3% of the coding region. The size of intron 1 for 5'-loxP site insertion: 3721 bp, and the size of intron 3 for 3'-loxP site insertion: 807 bp. The size of effective cKO region: ~2212 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Osr2 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(8712bp) | A(25.41% 2214) | C(24.77% 2158) | T(26.54% 2312) | G(23.28% 2028)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr15 + 35297051 35300050 3000 browser details YourSeq 42 1495 1539 3000 90.7% chr7 + 46670850 46670892 43 browser details YourSeq 38 1502 1539 3000 100.0% chr14 + 117853654 117853691 38 browser details YourSeq 35 1499 1535 3000 97.3% chr1 + 59177221 59177257 37 browser details YourSeq 29 150 179 3000 100.0% chr4 + 77819662 77819694 33 browser details YourSeq 25 1051 1075 3000 100.0% chr8 + 57741711 57741735 25 browser details YourSeq 23 70 92 3000 100.0% chr16 + 25336326 25336348 23 browser details YourSeq 21 2268 2288 3000 100.0% chr16 - 44775786 44775806 21

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% chr15 + 35302263 35305262 3000 browser details YourSeq 106 2375 2490 3000 99.2% chr1 + 171024774 171025177 404 browser details YourSeq 90 2378 2483 3000 97.1% chr16 + 10088008 10088176 169 browser details YourSeq 76 2410 2492 3000 96.4% chr4 - 11427095 11427274 180 browser details YourSeq 71 2417 2503 3000 96.3% chr11 - 84923842 84924361 520 browser details YourSeq 70 2376 2478 3000 82.7% chr13 - 42637717 42637792 76 browser details YourSeq 69 2419 2490 3000 98.7% chr12 + 97125837 97125910 74 browser details YourSeq 68 2418 2498 3000 93.6% chr19 - 59558432 59558582 151 browser details YourSeq 64 2423 2489 3000 98.5% chr2 + 53987987 53988073 87 browser details YourSeq 64 2376 2444 3000 97.2% chr10 + 78514796 78514866 71 browser details YourSeq 62 2419 2490 3000 98.6% chr2 - 33163686 33163819 134 browser details YourSeq 61 2418 2496 3000 95.6% chr1 + 190878542 190878636 95 browser details YourSeq 60 2380 2461 3000 93.1% chr6 + 144242906 144243024 119 browser details YourSeq 59 2419 2490 3000 86.2% chr2 - 168308038 168308103 66 browser details YourSeq 59 2419 2490 3000 97.0% chr1 + 132343963 132344076 114 browser details YourSeq 58 2417 2489 3000 97.0% chr2 - 120271587 120271701 115 browser details YourSeq 58 2419 2487 3000 87.1% chr14 - 87892653 87892715 63 browser details YourSeq 58 2418 2490 3000 88.3% chr7 + 132057871 132057941 71 browser details YourSeq 57 2419 2490 3000 87.4% chr14 - 3046126 3046193 68 browser details YourSeq 57 2418 2486 3000 92.8% chr12 + 72218726 72218810 85

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: Osr2 odd-skipped related 2 [ Mus musculus (house mouse) ] Gene ID: 107587, updated on 24-Oct-2019

Gene summary

Official Symbol Osr2 provided by MGI Official Full Name odd-skipped related 2 provided by MGI Primary source MGI:MGI:1930813 See related Ensembl:ENSMUSG00000022330 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 Osr2A; Osr2B; 5430409I15Rik Expression Biased expression in ovary adult (RPKM 68.7), subcutaneous fat pad adult (RPKM 65.0) and 10 other tissues See more Orthologs human all

Genomic context

Location: 15 B3.1; 15 14.46 cM See Osr2 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (35296098..35303305)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (35225867..35233060)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 5 transcripts

Gene: Osr2 ENSMUSG00000022330

Description odd-skipped related 2 [Source:MGI Symbol;Acc:MGI:1930813] Gene Synonyms 5430409I15Rik, Osr2A, Osr2B Location Chromosome 15: 35,296,098-35,303,305 forward strand. GRCm38:CM001008.2 About this gene This gene has 5 transcripts (splice variants), 186 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 15 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Osr2-201 ENSMUST00000022952.5 1724 276aa ENSMUSP00000022952.4 Protein coding CCDS27422 Q91ZD1 TSL:1 GENCODE basic

Osr2-204 ENSMUST00000228152.1 1324 312aa ENSMUSP00000154286.1 Protein coding - Q91ZD1 GENCODE basic APPRIS P1

Osr2-203 ENSMUST00000227655.1 2761 No protein - Retained intron - - -

Osr2-202 ENSMUST00000226838.1 1231 No protein - lncRNA - - -

Osr2-205 ENSMUST00000228684.1 579 No protein - lncRNA - - -

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27.21 kb Forward strand 35.29Mb 35.30Mb 35.31Mb Genes Osr2-201 >protein coding (Comprehensive set...

Osr2-203 >retained intron

Osr2-202 >lncRNA Osr2-204 >protein coding

Osr2-205 >lncRNA

Contigs AC116596.3 > Genes < BC048602-201lncRNA (Comprehensive set...

< BC048602-203lncRNA

< BC048602-202lncRNA

Regulatory Build

35.29Mb 35.30Mb 35.31Mb Reverse strand 27.21 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000022952

7.21 kb Forward strand

Osr2-201 >protein coding

ENSMUSP00000022... Low complexity (Seg) Superfamily Zinc finger C2H2 superfamily SMART Zinc finger C2H2-type Pfam Zinc finger C2H2-type PROSITE profiles Zinc finger C2H2-type PROSITE patterns Zinc finger C2H2-type PANTHER PTHR14196

PTHR14196:SF4 Gene3D 3.30.160.60 CDD cd05643

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 276

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

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