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Mouse Ripk3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ripk3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ripk3 gene (NCBI Reference Sequence: NM_001164108 ; Ensembl: ENSMUSG00000022221 ) is located on Mouse chromosome 14. 10 exons are identified, with the ATG start codon in exon 3 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000168716). Exon 3~9 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ripk3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-66O7 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 knock-out alleles exhibit resistance to induced inflammatory responses.

Exon 3 starts from about 100% of the coding region. The knockout of Exon 3~9 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 104 bp, and the size of intron 9 for 3'-loxP site insertion: 510 bp. The size of effective cKO region: ~2574 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 5 6 7 8 9 10 1 2 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Ripk3 cKO region Exon of mouse Adcy4 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8876bp) | A(24.93% 2213) | C(26.05% 2312) | T(24.04% 2134) | G(24.98% 2217)

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% chr14 - 55788186 55791185 3000 browser details YourSeq 118 1703 1903 3000 88.9% chr1 + 157720708 157720913 206 browser details YourSeq 108 1715 1895 3000 95.0% chr3 - 54165645 54165829 185 browser details YourSeq 104 1703 1893 3000 92.7% chr7 + 117417415 117417617 203 browser details YourSeq 96 1715 1903 3000 87.5% chr1 - 107784133 107784314 182 browser details YourSeq 87 1735 1899 3000 96.8% chr2 - 91191108 91191631 524 browser details YourSeq 84 607 922 3000 85.6% chr4 - 57277872 57278371 500 browser details YourSeq 80 1837 2163 3000 98.8% chr16 - 28759186 28759726 541 browser details YourSeq 75 1807 1899 3000 93.2% chr1 - 173821244 173821343 100 browser details YourSeq 73 1582 1904 3000 97.5% chr10 - 8486606 8487261 656 browser details YourSeq 68 1829 1899 3000 98.6% chr12 + 71043014 71043085 72 browser details YourSeq 64 1836 1899 3000 100.0% chr16 - 75257555 75257618 64 browser details YourSeq 64 1837 1903 3000 98.5% chr4 + 46850791 46850863 73 browser details YourSeq 64 572 688 3000 82.0% chr1 + 151670936 151671052 117 browser details YourSeq 61 1837 1905 3000 98.5% chr12 + 113735039 113735119 81 browser details YourSeq 60 1837 1899 3000 98.4% chr16 - 6157746 6157812 67 browser details YourSeq 60 1840 1899 3000 100.0% chr1 - 98876734 98876793 60 browser details YourSeq 59 1838 1896 3000 100.0% chr10 - 105142054 105142112 59 browser details YourSeq 58 1837 1894 3000 100.0% chr15 - 80184236 80184293 58 browser details YourSeq 58 1825 1885 3000 98.4% chr10 + 71652147 71652215 69

Note: The 3000 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 3000 1 3000 3000 100.0% chr14 - 55782612 55785611 3000 browser details YourSeq 48 677 881 3000 65.5% chr9 + 108073978 108074051 74 browser details YourSeq 35 2936 3000 3000 94.9% chr7 - 118810078 118810147 70 browser details YourSeq 28 2971 3000 3000 96.7% chrX - 12892671 12892700 30 browser details YourSeq 28 2507 2537 3000 96.7% chr7 - 87230839 87230871 33 browser details YourSeq 28 678 708 3000 96.7% chr2 - 142532350 142532399 50 browser details YourSeq 28 1059 1086 3000 100.0% chr1 + 32967389 32967416 28 browser details YourSeq 27 1055 1084 3000 96.6% chr16 - 40460229 40460258 30 browser details YourSeq 26 2491 2520 3000 96.6% chr14 + 120327801 120327834 34 browser details YourSeq 25 2970 3000 3000 90.4% chr16 - 5585666 5585696 31 browser details YourSeq 25 1165 1189 3000 100.0% chr11 - 65222421 65222445 25 browser details YourSeq 24 2973 3000 3000 92.9% chr6 - 73597171 73597198 28 browser details YourSeq 23 2976 3000 3000 96.0% chr8 + 121773026 121773050 25 browser details YourSeq 23 1056 1080 3000 87.5% chr15 + 37225665 37225688 24 browser details YourSeq 22 241 262 3000 100.0% chr13 - 16563676 16563697 22 browser details YourSeq 22 2975 2996 3000 100.0% chr1 + 69646311 69646332 22 browser details YourSeq 21 680 700 3000 100.0% chr5 - 116853506 116853526 21 browser details YourSeq 21 2934 2954 3000 100.0% chr5 - 4059341 4059361 21

Note: The 3000 bp section downstream of Exon 9 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ripk3 receptor-interacting serine-threonine kinase 3 [ Mus musculus (house mouse) ] Gene ID: 56532, updated on 22-Oct-2019

Gene summary

Official Symbol Ripk3 provided by MGI Official Full Name receptor-interacting serine-threonine kinase 3 provided by MGI Primary source MGI:MGI:2154952 See related Ensembl:ENSMUSG00000022221 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 Rip3; AW107945; 2610528K09Rik Annotation Note: Mprip (Gene ID: 26936) and Ripk3 (Gene ID: 56532) share the RIP3 symbol/alias in common. RIP3 is a widely used information alternative name for Ripk3 (Gene ID: 56532), which can be confused with Mprip (Gene ID: 26936), which also uses RIP3 as Expression an alternative name. [05 Mar 2019] Orthologs Broad expression in large intestine adult (RPKM 17.7), thymus adult (RPKM 10.3) and 18 other tissuesS ee more human all

Genomic context

Location: 14; 14 C3 See Ripk3 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (55784995..55788857, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (56403832..56407694, complement)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 9 transcripts

Gene: Ripk3 ENSMUSG00000022221

Description receptor-interacting serine-threonine kinase 3 [Source:MGI Symbol;Acc:MGI:2154952] Gene Synonyms 2610528K09Rik, Rip3 Location Chromosome 14: 55,784,995-55,788,865 reverse strand. GRCm38:CM001007.2 About this gene This gene has 9 transcripts (splice variants), 213 orthologues, 23 paralogues, 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

Ripk3- ENSMUST00000168716.7 1964 422aa ENSMUSP00000126306.1 Protein coding CCDS49499 E9PZP3 TSL:5 202 GENCODE basic

Ripk3- ENSMUST00000022830.13 1892 486aa ENSMUSP00000022830.6 Protein coding CCDS27131 Q9QZL0 TSL:1 201 GENCODE basic APPRIS P1

Ripk3- ENSMUST00000178399.2 1865 422aa ENSMUSP00000137278.1 Protein coding CCDS49499 E9PZP3 TSL:1 203 GENCODE basic

Ripk3- ENSMUST00000228326.1 658 158aa ENSMUSP00000154235.1 Protein coding - A0A2I3BQW9 CDS 3' incomplete 208

Ripk3- ENSMUST00000228476.1 482 160aa ENSMUSP00000153911.1 Protein coding - A0A2I3BPT3 CDS 5' and 3' 209 incomplete

Ripk3- ENSMUST00000228077.1 478 No - Retained - - - 206 protein intron

Ripk3- ENSMUST00000228175.1 383 No - Retained - - - 207 protein intron

Ripk3- ENSMUST00000227072.1 443 No - lncRNA - - - 205 protein

Ripk3- ENSMUST00000226203.1 360 No - lncRNA - - - 204 protein

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23.87 kb Forward strand 55.775Mb 55.780Mb 55.785Mb 55.790Mb 55.795Mb Contigs AC098877.3 > Genes (Comprehensive set... < Adcy4-202protein coding < Ripk3-201protein coding

< Adcy4-201protein coding < Ripk3-202protein coding

< Adcy4-203retained intron < Ripk3-206retained intron

< Adcy4-205protein codin

< Ripk3-203protein coding

< Ripk3-209protein coding

< Ripk3-207retained intron

< Ripk3-208protein coding

< Ripk3-204lncRNA

Regulatory Build

55.775Mb 55.780Mb 55.785Mb 55.790Mb 55.795Mb Reverse strand 23.87 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000168716

< Ripk3-202protein coding

Reverse strand 3.86 kb

ENSMUSP00000126... MobiDB lite Low complexity (Seg) Superfamily Protein kinase-like domain superfamily SMART Protein kinase domain Pfam Serine-threonine/tyrosine-protein kinase, catalytic domain RHIM domain

PROSITE profiles Protein kinase domain PROSITE patterns Serine/threonine-protein kinase, active site PIRSF PIRSF000654 PANTHER PTHR23257

PTHR23257:SF723 Gene3D 1.10.510.10

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

Variant Legend inframe insertion inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 422

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

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