https://www.alphaknockout.com Mouse Inava Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Inava conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Inava gene (NCBI Reference Sequence: NM_028872.3 ; Ensembl: ENSMUSG00000041605 ) is located on Mouse chromosome 1. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000120339). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Inava gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-100N3 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: Exon 2 starts from about 10.04% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 6240 bp, and the size of intron 2 for 3'-loxP site insertion: 609 bp. The size of effective cKO region: ~649 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 4 5 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Inava Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com 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(7149bp) | A(21.09% 1508) | C(26.47% 1892) | T(24.77% 1771) | G(27.67% 1978) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr1 - 136227913 136230912 3000 browser details YourSeq 37 1067 1105 3000 100.0% chr10 + 25482166 25482212 47 browser details YourSeq 24 1009 1040 3000 87.5% chr19 - 4212765 4212796 32 Note: The 3000 bp section upstream of Exon 2 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% chr1 - 136224264 136227263 3000 browser details YourSeq 25 103 129 3000 88.5% chr8 + 83831043 83831068 26 browser details YourSeq 23 109 131 3000 100.0% chr7 + 115074554 115074576 23 browser details YourSeq 22 109 130 3000 100.0% chr8 + 104796210 104796231 22 browser details YourSeq 22 109 130 3000 100.0% chr5 + 70174206 70174227 22 browser details YourSeq 21 110 130 3000 100.0% chr19 - 36082506 36082526 21 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Inava innate immunity activator [ Mus musculus (house mouse) ] Gene ID: 67313, updated on 26-Jun-2020 Gene summary Official Symbol Inava provided by MGI Official Full Name innate immunity activator provided by MGI Primary source MGI:MGI:1921579 See related Ensembl:ENSMUSG00000041605 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 D1Mgi54; AI586180; 1700034M08Rik; 4933426C09Rik; 5730559C18Rik Expression Biased expression in colon adult (RPKM 37.5), stomach adult (RPKM 18.3) and 11 other tissues See more Orthologs human all Genomic context Location: 1 E4; 1 See Inava in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (136213522..136234293, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (138110108..138130841, complement) Chromosome 1 - NC_000067.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Inava ENSMUSG00000041605 Description innate immunity activator [Source:MGI Symbol;Acc:MGI:1921579] Gene Synonyms 5730559C18Rik Location Chromosome 1: 136,213,531-136,234,264 reverse strand. GRCm38:CM000994.2 About this gene This gene has 6 transcripts (splice variants), 329 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Inava- ENSMUST00000120339.7 2988 677aa ENSMUSP00000113785.1 Protein coding CCDS15325 G3X9Z8 TSL:1 201 GENCODE basic APPRIS P1 Inava- ENSMUST00000144464.6 811 200aa ENSMUSP00000115554.1 Protein coding - D3YZT8 CDS 3' 202 incomplete TSL:5 Inava- ENSMUST00000150163.7 743 212aa ENSMUSP00000118074.1 Protein coding - D3YZL7 CDS 3' 203 incomplete TSL:3 Inava- ENSMUST00000195177.1 428 78aa ENSMUSP00000141506.1 Protein coding - A0A0A6YWD6 CDS 3' 206 incomplete TSL:3 Inava- ENSMUST00000153910.1 482 97aa ENSMUSP00000120263.1 Nonsense mediated - D6RJ44 TSL:3 204 decay Inava- ENSMUST00000194374.1 2387 No - Retained intron - - TSL:2 205 protein Page 6 of 8 https://www.alphaknockout.com 40.73 kb Forward strand 136.21Mb 136.22Mb 136.23Mb 136.24Mb Genes Gm26568-201 >antisense (Comprehensive set... Contigs AC124550.4 > Genes (Comprehensive set... < Mroh3-205protein coding < Inava-201protein coding < Mroh3-204nonsense mediated decay < Inava-203protein coding < Inava-202protein coding < Inava-205retained intron < Inava-204nonsense mediated decay < Inava-206protein coding Regulatory Build 136.21Mb 136.22Mb 136.23Mb 136.24Mb Reverse strand 40.73 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000120339 < Inava-201protein coding Reverse strand 20.73 kb ENSMUSP00000113... MobiDB lite Low complexity (Seg) Pfam Domain of unknown function DUF3338 PANTHER PTHR16093 Innate immunity activator protein All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 677 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.