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Mouse Antxr2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Antxr2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Antxr2 (NCBI Reference Sequence: NM_133738 ; Ensembl: ENSMUSG00000029338 ) is located on Mouse 5. 17 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 17 (Transcript: ENSMUST00000031281). Exon 8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Antxr2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-26L24 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 null mutations display female infertility. Mice homozygous for one allele are highly resistant to Bacillus anthracis or anthrax toxin induced lethality. Young mice homozygous for a second allele display pregnancy- related premature death and failure of parturition.

Exon 8 starts from about 43.6% of the coding region. The knockout of Exon 8 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 23192 bp, and the size of intron 8 for 3'-loxP site insertion: 2274 bp. The size of effective cKO region: ~561 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 8 17 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Antxr2 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(7061bp) | A(26.57% 1876) | C(19.37% 1368) | T(33.73% 2382) | G(20.32% 1435)

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% chr5 - 97980285 97983284 3000 browser details YourSeq 22 1356 1380 3000 95.9% chr5 - 29169331 29169356 26

Note: The 3000 bp section upstream of Exon 8 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% chr5 - 97976724 97979723 3000 browser details YourSeq 178 2764 2972 3000 91.4% chr7 + 28867985 28868182 198 browser details YourSeq 176 2740 2950 3000 95.9% chr11 + 97216174 97304500 88327 browser details YourSeq 168 2768 2956 3000 93.3% chr18 - 21606671 21606849 179 browser details YourSeq 166 2739 2964 3000 89.9% chr10 - 40426579 40426785 207 browser details YourSeq 166 2764 2988 3000 91.4% chr1 - 96382782 96383018 237 browser details YourSeq 165 2737 2952 3000 94.2% chr1 + 76678016 76678256 241 browser details YourSeq 163 2765 2964 3000 92.5% chr4 - 11569113 11569311 199 browser details YourSeq 159 2756 2956 3000 91.7% chr18 - 34100624 34100829 206 browser details YourSeq 159 2764 2988 3000 87.4% chr11 - 39632708 39632917 210 browser details YourSeq 159 2763 2974 3000 89.2% chr5 + 55516161 55516362 202 browser details YourSeq 157 2751 2938 3000 92.6% chr16 - 18523079 18523271 193 browser details YourSeq 156 2743 2928 3000 91.0% chr4 - 152091951 152092129 179 browser details YourSeq 154 2763 2973 3000 88.4% chr7 - 46824120 46824322 203 browser details YourSeq 154 2757 2983 3000 86.2% chr18 + 36714927 36715124 198 browser details YourSeq 152 2764 2947 3000 91.0% chr7 - 127067890 127068062 173 browser details YourSeq 152 2367 2943 3000 82.1% chr10 + 51575858 51576053 196 browser details YourSeq 151 2737 2920 3000 90.8% chr19 - 47725264 47725430 167 browser details YourSeq 150 2763 2956 3000 88.9% chr19 - 37135689 37135866 178 browser details YourSeq 150 2755 2943 3000 93.2% chr10 - 50706741 50706937 197

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Antxr2 anthrax toxin receptor 2 [ Mus musculus (house mouse) ] Gene ID: 71914, updated on 12-Aug-2019

Gene summary

Official Symbol Antxr2 provided by MGI Official Full Name anthrax toxin receptor 2 provided by MGI Primary source MGI:MGI:1919164 See related Ensembl:ENSMUSG00000029338 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 CMG2; CMG-2; cI-35; AW561899; 2310046B19Rik Expression Broad expression in heart adult (RPKM 13.0), subcutaneous fat pad adult (RPKM 7.8) and 24 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 E3 See Antxr2 in Genome Data Viewer

Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (97884688..98031017, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (98313707..98459981, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 3 transcripts

Gene: Antxr2 ENSMUSG00000029338

Description anthrax toxin receptor 2 [Source:MGI Symbol;Acc:MGI:1919164] Gene Synonyms 2310046B19Rik, CMG-2, CMG2, cI-35 Location Chromosome 5: 97,882,783-98,031,043 reverse strand. GRCm38:CM000998.2 About this gene This gene has 3 transcripts (splice variants), 250 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 14 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Antxr2- ENSMUST00000031281.13 5656 487aa ENSMUSP00000031281.9 Protein coding CCDS39177 Q3TCL6 Q6DFX2 TSL:1 201 GENCODE basic APPRIS P2

Antxr2- ENSMUST00000199088.1 2739 517aa ENSMUSP00000142605.1 Protein coding - A0A0G2JE26 TSL:1 202 GENCODE basic APPRIS ALT2

Antxr2- ENSMUST00000202736.1 545 No - lncRNA - - TSL:2 203 protein

168.26 kb Forward strand

97.90Mb 97.95Mb 98.00Mb Gm42759-201 >TEC (Comprehensive set...

Contigs AC125131.4 > < AC133519.3

Genes (Comprehensive set... < Antxr2-201protein coding

< Antxr2-202protein coding

< Antxr2-203lncRNA < C430019N01Rik-201TEC

Regulatory Build

97.90Mb 97.95Mb 98.00Mb Reverse strand 168.26 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

RNA gene processed transcript

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Transcript: ENSMUST00000031281

< Antxr2-201protein coding

Reverse strand 148.18 kb

ENSMUSP00000031... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily von Willebrand factor A-like domain superfamily SMART von Willebrand factor, type A Pfam von Willebrand factor, type A Anthrax toxin receptor, extracellular domain Anthrax toxin receptor, C-terminal

PROSITE profiles von Willebrand factor, type A PIRSF Anthrax toxin receptor PANTHER Anthrax toxin receptor

PTHR16059 Gene3D von Willebrand factor A-like domain superfamily CDD cd01474

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 487

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

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