https://www.alphaknockout.com Mouse Cabin1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cabin1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cabin1 gene (NCBI Reference Sequence: NM_172549 ; Ensembl: ENSMUSG00000020196 ) is located on Mouse chromosome 10. 37 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 37 (Transcript: ENSMUST00000001712). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cabin1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-7G24 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: Homozygous null mutants exhibit embryonic lethality during organogenesis. Mice producing a truncated protein exhibit elevated levels of serum IgG1, IgG2b and IgE, produce more IgG1 in response to T-cell dependent antigen, and have enhanced expression of cytokines in response to anti-CD3 stimulation. Exon 5 starts from about 3.23% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 739 bp, and the size of intron 6 for 3'-loxP site insertion: 1262 bp. The size of effective cKO region: ~2576 bp. These transcripts Cabin1-203, Cabin1-204 and Cabin1-212 may not be affected by deleting this cKO region. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 37 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Cabin1 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(9076bp) | A(22.61% 2052) | C(22.26% 2020) | T(30.23% 2744) | G(24.9% 2260) 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. 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% chr10 - 75753762 75756761 3000 browser details YourSeq 40 2530 2908 3000 57.8% chr2 + 172611335 172611464 130 browser details YourSeq 38 12 61 3000 93.1% chr4 + 62353368 62353417 50 browser details YourSeq 37 12 52 3000 97.5% chr2 - 26316856 26316896 41 browser details YourSeq 34 11 51 3000 84.7% chr4 - 67762943 67762981 39 browser details YourSeq 34 25 61 3000 97.3% chr2 - 49718084 49718122 39 browser details YourSeq 34 2706 2769 3000 69.5% chr1 + 179482813 179482871 59 browser details YourSeq 32 12 47 3000 97.1% chr2 - 30921671 30921708 38 browser details YourSeq 32 2689 2727 3000 88.9% chr18 - 34670709 34670746 38 browser details YourSeq 32 25 56 3000 100.0% chr14 + 7807090 7807121 32 browser details YourSeq 31 25 65 3000 91.9% chr7 + 127806270 127806316 47 browser details YourSeq 30 24 56 3000 87.1% chr11 - 106750341 106750371 31 browser details YourSeq 30 18 47 3000 100.0% chrX + 97133907 97133936 30 browser details YourSeq 30 88 117 3000 100.0% chr1 + 169731535 169731564 30 browser details YourSeq 29 22 51 3000 100.0% chr7 - 131714021 131714052 32 browser details YourSeq 29 25 56 3000 96.9% chr1 - 65385191 65385224 34 browser details YourSeq 28 27 55 3000 100.0% chr2 - 170678209 170678273 65 browser details YourSeq 27 25 55 3000 93.6% chr15 - 70510531 70510561 31 browser details YourSeq 27 29 61 3000 96.7% chr10 - 117689556 117689588 33 browser details YourSeq 27 439 471 3000 91.0% chr10 + 39208046 39208078 33 Note: The 3000 bp section upstream of Exon 5 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% chr10 - 75748186 75751185 3000 browser details YourSeq 346 1838 2260 3000 90.2% chr14 - 24719086 24719496 411 browser details YourSeq 343 1838 2256 3000 90.9% chr10 - 63563114 63563524 411 browser details YourSeq 342 1838 2265 3000 89.8% chr1 - 150376864 150377278 415 browser details YourSeq 338 1838 2260 3000 90.6% chr7 - 129277219 129277633 415 browser details YourSeq 334 1838 2252 3000 91.0% chr19 + 14557180 14557585 406 browser details YourSeq 332 1842 2360 3000 89.6% chr14 - 101533734 101534355 622 browser details YourSeq 331 1838 2250 3000 90.7% chr5 - 21284143 21284543 401 browser details YourSeq 331 1838 2254 3000 89.7% chr16 + 6082673 6083077 405 browser details YourSeq 331 1840 2264 3000 92.0% chr13 + 6553399 6554066 668 browser details YourSeq 330 1838 2254 3000 92.0% chr8 + 110323646 110324065 420 browser details YourSeq 329 1838 2237 3000 90.9% chr11 + 87251714 87252101 388 browser details YourSeq 327 1838 2247 3000 91.4% chr1 - 43638396 43638809 414 browser details YourSeq 326 1838 2250 3000 89.0% chrX - 113388180 113388579 400 browser details YourSeq 326 1838 2250 3000 89.4% chr7 + 92459928 92460331 404 browser details YourSeq 326 1838 2251 3000 89.4% chr17 + 88940088 88940490 403 browser details YourSeq 325 1838 2252 3000 90.7% chr5 - 40703957 40704370 414 browser details YourSeq 325 1838 2252 3000 91.2% chr15 - 58923103 58923505 403 browser details YourSeq 324 1838 2248 3000 91.7% chr3 - 117476168 117476564 397 browser details YourSeq 323 1838 2250 3000 89.0% chr3 + 101772911 101773313 403 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Cabin1 calcineurin binding protein 1 [ Mus musculus (house mouse) ] Gene ID: 104248, updated on 12-Aug-2019 Gene summary Official Symbol Cabin1 provided by MGI Official Full Name calcineurin binding protein 1 provided by MGI Primary source MGI:MGI:1298375 See related Ensembl:ENSMUSG00000020196 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 Cain; Ppp3in; A330070M20Rik Expression Ubiquitous expression in thymus adult (RPKM 14.2), ovary adult (RPKM 14.2) and 28 other tissues See more Orthologs human all Genomic context Location: 10 C1; 10 38.56 cM See Cabin1 in Genome Data Viewer Exon count: 37 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (75646110..75764357, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (75108855..75227102, complement) Chromosome 10 - NC_000076.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 13 transcripts Gene: Cabin1 ENSMUSG00000020196 Description calcineurin binding protein 1 [Source:MGI Symbol;Acc:MGI:1298375] Gene Synonyms A330070M20Rik, Cain, Ppp3in Location Chromosome 10: 75,646,112-75,764,341 reverse strand. GRCm38:CM001003.2 About this gene This gene has 13 transcripts (splice variants), 209 orthologues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cabin1- ENSMUST00000001712.7 7417 2187aa ENSMUSP00000001712.6 Protein coding CCDS35938 G3X8Q1 TSL:5 201 GENCODE basic APPRIS P1 Cabin1- ENSMUST00000217810.1 2115 473aa ENSMUSP00000152043.1 Protein coding - A0A1W2P8C4 CDS 5' 203 incomplete TSL:1 Cabin1- ENSMUST00000219806.1 573 191aa ENSMUSP00000151879.1 Protein coding - A0A1W2P819 CDS 5' and 3' 212 incomplete TSL:3 Cabin1- ENSMUST00000218469.1 361 18aa ENSMUSP00000151933.1 Protein coding - A0A1W2P8A1 CDS 3' 204 incomplete TSL:3 Cabin1- ENSMUST00000218878.1 705 32aa ENSMUSP00000151485.1 Nonsense mediated - A0A1W2P755 CDS 5' 209 decay incomplete TSL:5 Cabin1- ENSMUST00000218790.1 593 39aa ENSMUSP00000151436.1 Nonsense mediated - A0A1W2P6X4 CDS 5' 207 decay incomplete TSL:3 Cabin1- ENSMUST00000218480.1 3095 No - Retained intron - - TSL:NA 205 protein Cabin1- ENSMUST00000220009.1 854 No - Retained intron - - TSL:3 213 protein Cabin1- ENSMUST00000218486.1 826 No - Retained intron - - TSL:3 206 protein Cabin1- ENSMUST00000218951.1 491 No - Retained intron - - TSL:2 210 protein Cabin1- ENSMUST00000218814.1 718 No - lncRNA - - TSL:2 208 protein Cabin1- ENSMUST00000219682.1 634 No - lncRNA - - TSL:3 211 protein Cabin1- ENSMUST00000217664.1 341 No - lncRNA - - TSL:3 202 protein Page 6 of 8 https://www.alphaknockout.com 138.23 kb Forward strand 75.64Mb 75.66Mb 75.68Mb 75.70Mb 75.72Mb 75.74Mb 75.76Mb Genes Gm19326-201 >processed pseudogene (Comprehensive set... Contigs AC087540.34 > < AC142499.9 Genes (Comprehensive set... < Susd2-202protein