Mouse Rnf114 Knockout Project (CRISPR/Cas9)

http://www.alphaknockout.com/ Mouse Rnf114 Knockout Project (CRISPR/Cas9)

Objective: To create a Rnf114 knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rnf114 gene ( NCBI Reference Sequence: NM_030743 ; Ensembl: ENSMUSG00000006418 ) is located on mouse chromosome 2. 8 exons are identified , with the ATG start codon in exon 3 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000109214). Exon 3~8 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:

Exon 3 starts from the coding region. Exon 3~8 covers 100.0% of the coding region. The size of effective KO region: ~12205 bp.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 3 4 5 6 7 8

Legends Exon of mouse Rnf114 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 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 8 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(26.75% 535) | C(26.1% 522) | G(24.25% 485) | T(22.9% 458)

Note: The 2000 bp section upstream 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(23.75% 475) | C(26.0% 520) | G(24.95% 499) | T(25.3% 506)

Note: The 2000 bp section downstream of Exon 8 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 2000 1 2000 2000 100.0% chr2 + 167501438 167503437 2000 browser details YourSeq 204 640 949 2000 89.9% chr11 + 52052155 52082746 30592 browser details YourSeq 194 661 952 2000 91.5% chr3 + 89055827 89056406 580 browser details YourSeq 161 682 924 2000 87.6% chr5 - 142980296 142980833 538 browser details YourSeq 146 596 941 2000 86.2% chr1 - 60145880 60146544 665 browser details YourSeq 130 605 1061 2000 82.3% chr15 - 58908208 58908558 351 browser details YourSeq 128 698 976 2000 91.2% chr11 + 70443989 70444511 523 browser details YourSeq 124 233 776 2000 83.0% chr1 + 133095749 133096093 345 browser details YourSeq 122 651 915 2000 91.3% chrX - 85711663 85712078 416 browser details YourSeq 122 602 951 2000 79.9% chr17 - 8598757 8598940 184 browser details YourSeq 117 641 792 2000 92.1% chr3 - 138650366 138650781 416 browser details YourSeq 117 641 776 2000 94.7% chr11 - 89323891 89324026 136 browser details YourSeq 117 609 954 2000 78.1% chr12 + 71305592 71305771 180 browser details YourSeq 116 602 953 2000 78.7% chr17 - 23622835 23623031 197 browser details YourSeq 115 597 776 2000 90.8% chr18 - 23580818 23581180 363 browser details YourSeq 114 813 958 2000 85.8% chr11 + 70927602 70927741 140 browser details YourSeq 113 596 773 2000 93.1% chr8 - 33650349 33650891 543 browser details YourSeq 113 813 968 2000 85.8% chr1 - 133697325 133697477 153 browser details YourSeq 113 818 969 2000 86.1% chr16 + 44071435 44071581 147 browser details YourSeq 112 641 776 2000 93.8% chr6 - 83968400 83968536 137

Note: The 2000 bp section upstream of Exon 3 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 2000 1 2000 2000 100.0% chr2 + 167516168 167518167 2000 browser details YourSeq 37 745 791 2000 95.0% chr6 - 129187841 129187887 47 browser details YourSeq 36 747 792 2000 97.4% chr7 - 36457339 36457385 47 browser details YourSeq 35 745 792 2000 94.9% chr13 - 24046090 24046140 51 browser details YourSeq 33 748 791 2000 94.6% chr7 - 80663819 80663863 45 browser details YourSeq 32 749 783 2000 97.2% chr9 - 45034275 45034310 36 browser details YourSeq 32 752 792 2000 92.5% chr5 - 110014956 110015002 47 browser details YourSeq 32 745 786 2000 97.1% chr4 - 128941300 128941342 43 browser details YourSeq 32 745 781 2000 94.3% chr4 + 124813660 124813697 38 browser details YourSeq 31 49 85 2000 97.1% chrX + 5561041 5561087 47 browser details YourSeq 30 755 790 2000 96.9% chr9 - 18708741 18708777 37 browser details YourSeq 30 1935 1974 2000 87.5% chr17 - 57164719 57164758 40 browser details YourSeq 30 1935 1974 2000 87.5% chr17 - 28514659 28514698 40 browser details YourSeq 30 747 792 2000 89.5% chr11 - 70665735 70665783 49 browser details YourSeq 30 745 781 2000 94.0% chr9 + 101255151 101255187 37 browser details YourSeq 30 745 791 2000 94.2% chr4 + 124289156 124289204 49 browser details YourSeq 29 746 791 2000 94.0% chr4 - 147897776 147897821 46 browser details YourSeq 29 1935 1973 2000 87.2% chr14 - 120485175 120485213 39 browser details YourSeq 29 726 762 2000 87.1% chr9 + 113247459 113247493 35 browser details YourSeq 28 1935 1976 2000 83.4% chr15 - 89492645 89492686 42

Note: The 2000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 http://www.alphaknockout.com/ Gene and protein information: Rnf114 ring finger protein 114 [ Mus musculus (house mouse) ] Gene ID: 81018, updated on 17-Nov-2020

Gene summary

Official Symbol Rnf114 provided by MGI Official Full Name ring finger protein 114 provided by MGI Primary source MGI:MGI:1933159 See related Ensembl:ENSMUSG00000006418 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 Zfp31; Znf22; Zfp228; Zfp313; Znf228; AI225886; AW549494; 1110008J21Rik Expression Ubiquitous expression in thymus adult (RPKM 45.5), large intestine adult (RPKM 37.9) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 H3 See Rnf114 in Genome Data Viewer Exon count: 8

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) 2 NC_000068.8 (167334565..167358093)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (167492645..167516173)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (167318145..167341666)

Chromosome 2 - NC_000068.8

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Transcript information: This gene has 3 transcripts

Gene: Rnf114 ENSMUSG00000006418

Description ring finger protein 114 [Source:MGI Symbol;Acc:MGI:1933159] Gene Synonyms 1110008J21Rik, Zfp313, Znf228 Location Chromosome 2: 167,492,645-167,516,173 forward strand. GRCm38:CM000995.2 About this gene This gene has 3 transcripts (splice variants), 291 orthologues and 5 paralogues. Transcripts

UniProt Name Transcript ID bp Protein Translation ID Biotype CCDS Flags Match

Rnf114- ENSMUST00000109214.7 3001 229aa ENSMUSP00000104837.1 Protein coding CCDS17101 Q9ET26 TSL:1 202 GENCODE basic APPRIS P1

Rnf114- ENSMUST00000078050.6 2502 229aa ENSMUSP00000077197.6 Protein coding CCDS17101 Q9ET26 TSL:1 201 GENCODE basic APPRIS P1

Rnf114- ENSMUST00000127939.1 1985 57aa ENSMUSP00000138430.1 Nonsense mediated - S4R1Z0 TSL:1 203 decay

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43.53 kb Forward strand 167.49Mb 167.50Mb 167.51Mb 167.52Mb Genes (Comprehensive set... Rnf114-202 >protein coding

Rnf114-201 >protein coding

Rnf114-203 >nonsense mediated decay

Contigs AL589870.30 >

Genes < Spata2-201protein coding (Comprehensive set...

< Spata2-202protein coding

< Spata2-204retained intron

< Spata2-203processed transcript

< Spata2-206processed transcript

< Spata2-205processed transcript

Regulatory Build

167.49Mb 167.50Mb 167.51Mb 167.52Mb Reverse strand 43.53 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

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Transcript: ENSMUST00000109214

23.52 kb Forward strand

Rnf114-202 >protein coding

ENSMUSP00000104... MobiDB lite Low complexity (Seg) Superfamily SSF57850

SMART Zinc finger, RING-type Zinc finger C2H2-type

Pfam Zinc finger C2HC RNF-type Drought induced 19 protein type, zinc-binding domain

RING-type zinc-finger, LisH dimerisation motif PROSITE profiles Zinc finger, RING-type Zinc finger C2HC RNF-type

PROSITE patterns Zinc finger, RING-type, conserved site PANTHER PTHR46016:SF3

PTHR46016 Gene3D Zinc finger, RING/FYVE/PHD-type CDD E3 ubiquitin-protein ligase RNF114, RING finger, HC subclass

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

Variant Legend stop lost missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 200 229

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

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