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Mouse Relt Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Relt (NCBI Reference Sequence: NM_177073 ; Ensembl: ENSMUSG00000008318 ) is located on Mouse 7. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000008462). Exon 3~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Relt gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-98O8 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 with a premature termination codon resulting in a null allele exhibit incisor and molar enamel malformations.

Exon 3 starts from about 5.12% of the coding region. The knockout of Exon 3~5 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1331 bp, and the size of intron 5 for 3'-loxP site insertion: 770 bp. The size of effective cKO region: ~1122 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 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Relt 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(7622bp) | A(21.29% 1623) | C(24.43% 1862) | T(24.91% 1899) | G(29.36% 2238)

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% chr7 - 100851965 100854964 3000 browser details YourSeq 70 1848 1919 3000 98.7% chr7 - 25422727 25422798 72 browser details YourSeq 23 2620 2642 3000 100.0% chr6 + 65945186 65945208 23 browser details YourSeq 22 2165 2186 3000 100.0% chr18 - 62491216 62491237 22 browser details YourSeq 20 1113 1132 3000 100.0% chr1 + 37664909 37664928 20

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% chr7 - 100847843 100850842 3000 browser details YourSeq 473 520 2729 3000 91.5% chr7 - 25421866 25422405 540 browser details YourSeq 403 548 2821 3000 83.4% chrX + 154436060 154436704 645 browser details YourSeq 34 2761 2838 3000 73.7% chr7 - 100850432 100850493 62

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: Relt RELT tumor necrosis factor receptor [ Mus musculus (house mouse) ] Gene ID: 320100, updated on 10-Oct-2019

Gene summary

Official Symbol Relt provided by MGI Official Full Name RELT tumor necrosis factor receptor provided by MGI Primary source MGI:MGI:2443373 See related Ensembl:ENSMUSG00000008318 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 Tnfrsf19l; E430021K24Rik Expression Broad expression in spleen adult (RPKM 9.5), thymus adult (RPKM 6.5) and 24 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 E2 See Relt in Genome Data Viewer

Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (100845847..100863489, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (107994362..108011927, complement)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 6 transcripts

Gene: Relt ENSMUSG00000008318

Description RELT tumor necrosis factor receptor [Source:MGI Symbol;Acc:MGI:2443373] Gene Synonyms E430021K24Rik, Tnfrsf19l Location Chromosome 7: 100,845,847-100,863,446 reverse strand. GRCm38:CM001000.2 About this gene This gene has 6 transcripts (splice variants), 177 orthologues, 21 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Relt- ENSMUST00000008462.10 2851 436aa ENSMUSP00000008462.3 Protein coding CCDS21506 Q8BX43 TSL:1 201 GENCODE basic APPRIS P1

Relt- ENSMUST00000136231.1 516 119aa ENSMUSP00000121443.1 Protein coding - D3Z306 CDS 3' incomplete 202 TSL:2

Relt- ENSMUST00000139604.7 459 123aa ENSMUSP00000119208.1 Protein coding - D3YWL9 CDS 3' incomplete 203 TSL:5

Relt- ENSMUST00000156855.7 428 123aa ENSMUSP00000120042.1 Protein coding - D3YWL9 CDS 3' incomplete 206 TSL:5

Relt- ENSMUST00000142885.1 346 58aa ENSMUSP00000121049.1 Protein coding - D3YTN8 CDS 3' incomplete 204 TSL:3

Relt- ENSMUST00000155413.8 1966 119aa ENSMUSP00000118150.2 Nonsense mediated decay - D6RIM1 TSL:1 205

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37.60 kb Forward strand

100.84Mb 100.85Mb 100.86Mb 100.87Mb Fam168a-202 >protein coding (Comprehensive set...

Fam168a-204 >protein coding

Fam168a-201 >protein coding

Fam168a-205 >protein coding

Fam168a-206 >protein coding

Fam168a-210 >protein coding

Contigs AC150744.2 > Genes (Comprehensive set... < Relt-201protein coding < Arhgef17-201protein coding

< Relt-205nonsense mediated decay < Arhgef17-204protein coding

< Relt-203protein coding

< Relt-206protein coding

< Relt-202protein coding

< Relt-204protein coding

Regulatory Build

100.84Mb 100.85Mb 100.86Mb 100.87Mb Reverse strand 37.60 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000008462

< Relt-201protein coding

Reverse strand 17.60 kb

ENSMUSP00000008... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... SMART TNFR/NGFR cysteine-rich region

Prints Tumour necrosis factor receptor 19-like Pfam TNFR/NGFR cysteine-rich region TNF receptor family, RELT

PANTHER Tumour necrosis factor receptor 19-like

Gene3D 2.10.50.10

CDD Tumor necrosis factor receptor 19-like, N-terminal

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 436

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

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