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Mouse Rnf5 Knockout Project (CRISPR/Cas9)

Objective: To create a Rnf5 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rnf5 (NCBI Reference Sequence: NM_019403 ; Ensembl: ENSMUSG00000015478 ) is located on Mouse 17. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000015622). Exon 1~3 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: When subjected to muscle damage by cardiotoxin treatment, mice homozygous for a targeted null mutation display attenuated muscle regeneration associated with a delayed ER stress response.

Exon 1 starts from the coding region. Exon 1~3 covers 50.37% of the coding region. The size of effective KO region: ~1389 bp. The KO 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 6

Legends Exon of mouse Rnf5 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 1 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 96 bp section downstream of Exon 3 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(21.4% 428) | C(29.15% 583) | T(24.4% 488) | G(25.05% 501)

Note: The 2000 bp section upstream of Exon 1 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(96bp) | A(17.71% 17) | C(31.25% 30) | T(21.88% 21) | G(29.17% 28)

Note: The 96 bp section downstream of Exon 3 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 140 1 140 140 100.0% chr17 - 34603333 34603472 140 browser details YourSeq 99 35 139 140 95.1% chr9 - 120755383 120755485 103 browser details YourSeq 21 48 68 140 100.0% chr7 - 141193997 141194017 21 browser details YourSeq 21 115 135 140 100.0% chr7 - 98476852 98476872 21 browser details YourSeq 21 48 68 140 100.0% chr2 - 133552038 133552058 21

Note: The 140 bp section upstream of Exon 1 is BLAT searched against the genome.

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Gene and information: Rnf5 ring finger protein 5 [ Mus musculus (house mouse) ] Gene ID: 54197, updated on 10-Oct-2019

Gene summary

Official Symbol Rnf5 provided by MGI Official Full Name ring finger protein 5 provided by MGI Primary source MGI:MGI:1860076 See related Ensembl:ENSMUSG00000015478 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 NG2; AA407576; 2410131O05Rik Expression Ubiquitous expression in kidney adult (RPKM 88.4), large intestine adult (RPKM 73.5) and 28 other tissues See more Orthologs human all

Genomic context

Location: 17 B1; 17 18.18 cM See Rnf5 in Genome Data Viewer Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (34601099..34603561, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (34738044..34740506, complement)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 3 transcripts

Gene: Rnf5 ENSMUSG00000015478

Description ring finger protein 5 [Source:MGI Symbol;Acc:MGI:1860076] Gene Synonyms 2410131O05Rik Location Chromosome 17: 34,601,091-34,603,690 reverse strand. GRCm38:CM001010.2 About this gene This gene has 3 transcripts (splice variants), 164 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rnf5-201 ENSMUST00000015622.7 1238 180aa ENSMUSP00000015622.7 Protein coding CCDS28650 O35445 TSL:1 GENCODE basic APPRIS P1

Rnf5-203 ENSMUST00000174475.7 850 No protein - Retained intron - - TSL:2

Rnf5-202 ENSMUST00000174045.1 763 No protein - Retained intron - - TSL:2

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22.60 kb Forward strand 34.595Mb 34.600Mb 34.605Mb 34.610Mb Gpsm3-201 >protein coding Pbx2-202 >retained intron Gm20463-201 >lncRNA Agpat1-206 >protein coding (Comprehensive set...

Gpsm3-202 >nonsense mediated decay Ager-209 >protein coding Agpat1-204 >protein coding

Gpsm3-203 >protein coding Ager-201 >protein coding Agpat1-201 >protein coding

Pbx2-203 >protein coding Ager-212 >retained intron Agpat1-205 >nonsense mediated decay

Pbx2-201 >protein coding Ager-213 >lncRNA Agpat1-207 >nonsense mediated decay

Pbx2-205 >protein coding Ager-204 >retained intron Agpat1-202 >protein coding

Pbx2-204 >protein coding Ager-208 >nonsense mediated decay Agpat1-208 >protein coding

Ager-207 >protein coding Agpat1-203 >retained intron

Ager-206 >retained intron

Ager-202 >retained intron

Ager-210 >retained intron

Ager-205 >retained intron

Ager-211 >retained intron

Ager-203 >retained intron

Contigs CT009767.6 > Genes (Comprehensive set... < Rnf5-201protein coding < Egfl8-206retained intron

< Rnf5-203retained intron < Egfl8-202protein coding

< Rnf5-202retained intron < Egfl8-201protein coding

Regulatory Build

34.595Mb 34.600Mb 34.605Mb 34.610Mb Reverse strand 22.60 kb

Regulation Legend Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000015622

< Rnf5-201protein coding

Reverse strand 2.60 kb

ENSMUSP00000015... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily SSF57850 SMART Zinc finger, RING-type Pfam PF13920 PROSITE profiles Zinc finger, RING-type PROSITE patterns Zinc finger, RING-type, conserved site PANTHER PTHR12313:SF4

PTHR12313 Gene3D Zinc finger, RING/FYVE/PHD-type CDD cd16743

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

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

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