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Mouse Rnf24 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rnf24 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rnf24 (NCBI Reference Sequence: NM_178607 ; Ensembl: ENSMUSG00000048911 ) is located on Mouse 2. 6 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 6 (Transcript: ENSMUST00000059372). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rnf24 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-20L13 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:

Exon 4 starts from about 42.12% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2781 bp, and the size of intron 5 for 3'-loxP site insertion: 671 bp. The size of effective cKO region: ~1942 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 4 5 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rnf24 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(8442bp) | A(26.13% 2206) | C(22.32% 1884) | T(28.99% 2447) | G(22.57% 1905)

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 - 131305944 131308943 3000 browser details YourSeq 225 1931 2238 3000 92.8% chr10 - 128264355 128264664 310 browser details YourSeq 219 1931 2250 3000 91.7% chr4 + 82999571 82999903 333 browser details YourSeq 197 1679 2246 3000 86.4% chr11 + 98975636 98976155 520 browser details YourSeq 183 1931 2250 3000 89.0% chr2 + 126834584 126835190 607 browser details YourSeq 168 1949 2249 3000 92.9% chrX - 151043035 151043601 567 browser details YourSeq 166 1977 2251 3000 91.9% chr19 + 4277631 4278480 850 browser details YourSeq 135 2075 2250 3000 90.5% chr11 - 29234015 29234202 188 browser details YourSeq 135 1723 2247 3000 88.6% chr2 + 164406888 164407433 546 browser details YourSeq 131 2072 2244 3000 89.3% chr17 + 29528087 29528262 176 browser details YourSeq 127 2075 2250 3000 90.1% chr11 - 20609841 20610018 178 browser details YourSeq 112 1686 1830 3000 93.8% chr18 - 61166899 61167072 174 browser details YourSeq 109 2097 2239 3000 88.9% chr5 - 100482725 100482871 147 browser details YourSeq 108 1973 2417 3000 82.4% chr17 + 88197837 88198243 407 browser details YourSeq 106 2090 2234 3000 87.4% chr10 + 50065807 50065959 153 browser details YourSeq 104 1686 1819 3000 94.1% chr7 - 29904854 29905015 162 browser details YourSeq 104 1723 1989 3000 90.0% chr11 - 21361734 21362073 340 browser details YourSeq 104 1687 1832 3000 91.2% chr12 + 72949808 72949981 174 browser details YourSeq 102 1934 2049 3000 94.8% chr7 - 29905322 29905451 130 browser details YourSeq 102 2097 2221 3000 91.2% chr18 - 77085364 77085489 126

Note: The 3000 bp section upstream of Exon 4 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 - 131301002 131304001 3000 browser details YourSeq 1324 1336 2969 3000 92.5% chr3 + 30385341 30387153 1813 browser details YourSeq 25 2184 2217 3000 85.2% chrX + 107934001 107934032 32

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Rnf24 ring finger protein 24 [ Mus musculus (house mouse) ] Gene ID: 51902, updated on 12-Aug-2019

Gene summary

Official Symbol Rnf24 provided by MGI Official Full Name ring finger protein 24 provided by MGI Primary source MGI:MGI:1261771 See related Ensembl:ENSMUSG00000048911 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 C86507; AI317164; D2Ertd504e; 2810473M14Rik; 4930505A13Rik Expression Broad expression in testis adult (RPKM 10.0), whole brain E14.5 (RPKM 9.9) and 25 other tissues See more Orthologs human all

Genomic context

Location: 2 F1; 2 63.32 cM See Rnf24 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (131298488..131352892, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (131124224..131178628, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 5 transcripts

Gene: Rnf24 ENSMUSG00000048911

Description ring finger protein 24 [Source:MGI Symbol;Acc:MGI:1261771] Gene Synonyms 2810473M14Rik, 4930505A13Rik, C86507, D2Ertd504e Location Chromosome 2: 131,298,064-131,352,892 reverse strand. GRCm38:CM000995.2 About this gene This gene has 5 transcripts (splice variants), 194 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rnf24- ENSMUST00000059372.10 5978 148aa ENSMUSP00000058630.4 Protein coding CCDS16762 Q4VA59 TSL:1 201 Q8BGI1 GENCODE basic APPRIS P1

Rnf24- ENSMUST00000110194.7 5944 148aa ENSMUSP00000105823.1 Protein coding CCDS16762 Q4VA59 TSL:1 202 Q8BGI1 GENCODE basic APPRIS P1

Rnf24- ENSMUST00000165420.7 5833 148aa ENSMUSP00000129843.1 Protein coding CCDS16762 Q4VA59 TSL:3 204 Q8BGI1 GENCODE basic APPRIS P1

Rnf24- ENSMUST00000183902.1 1466 95aa ENSMUSP00000139130.1 Nonsense mediated - V9GXF9 TSL:1 205 decay

Rnf24- ENSMUST00000142982.1 913 No - Retained intron - - TSL:2 203 protein

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74.83 kb Forward strand 131.30Mb 131.32Mb 131.34Mb 131.36Mb Mir103-2-201 >miRNA Gm14233-201 >lncRNA (Comprehensive set...

Pank2-204 >protein coding

Pank2-207 >nonsense mediated decay

Pank2-208 >nonsense mediated decay

Pank2-203 >protein coding

Pank2-202 >lncRNA

Pank2-206 >nonsense mediated decay

Pank2-205 >nonsense mediated decay

Contigs AL808128.4 > Genes (Comprehensive set... < Rnf24-201protein coding

< Rnf24-204protein coding < Snrpert-201processed pseudogene

< Rnf24-202protein coding

< Rnf24-205nonsense mediated decay

< Rnf24-203retained intron

Regulatory Build

131.30Mb 131.32Mb 131.34Mb 131.36Mb Reverse strand 74.83 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript pseudogene RNA gene

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Transcript: ENSMUST00000059372

< Rnf24-201protein coding

Reverse strand 54.82 kb

ENSMUSP00000058... Transmembrane heli... Superfamily SSF57850

SMART Zinc finger, RING-type

Zinc finger, RING-CH-type Pfam Zinc finger, RING-type

PROSITE profiles Zinc finger, RING-type

PANTHER RING finger protein 24

PTHR22763 Gene3D Zinc finger, RING/FYVE/PHD-type

CDD cd16469

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

Variant Legend

synonymous variant

Scale bar 0 20 40 60 80 100 120 148

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

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