https://www.alphaknockout.com Mouse Card11 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Card11 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Card11 gene (NCBI Reference Sequence: NM_175362 ; Ensembl: ENSMUSG00000036526 ) is located on Mouse chromosome 5. 25 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 25 (Transcript: ENSMUST00000085786). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Card11 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-57O6 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 targeted null mutation exhibit defects in antigen receptor signalling in both T and B lymphocytes. Exon 4 starts from about 6.38% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 4545 bp, and the size of intron 5 for 3'-loxP site insertion: 3707 bp. The size of effective cKO region: ~2528 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 4 5 25 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Card11 Homology arm cKO region loxP site Page 2 of 7 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. 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(9028bp) | A(27.72% 2503) | C(22.09% 1994) | T(24.59% 2220) | G(25.6% 2311) 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 7 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% chr5 - 140908903 140911902 3000 browser details YourSeq 505 1 587 3000 92.8% chr5 - 139937075 139937658 584 browser details YourSeq 504 1 587 3000 93.7% chr7 - 19486078 19943100 457023 browser details YourSeq 500 1 586 3000 93.5% chr7 - 28638606 28884204 245599 browser details YourSeq 500 1 566 3000 94.4% chr7 + 30905424 30905991 568 browser details YourSeq 500 1 587 3000 93.7% chr5 + 144637824 144638459 636 browser details YourSeq 497 1 587 3000 93.1% chr7 + 19908284 19908927 644 browser details YourSeq 496 1 587 3000 92.8% chr7 + 18945663 19351235 405573 browser details YourSeq 495 1 577 3000 94.2% chr7 + 28202654 28706171 503518 browser details YourSeq 495 1 567 3000 94.0% chr5 + 139897995 139898564 570 browser details YourSeq 491 1 577 3000 92.9% chr5 + 137157735 137158312 578 browser details YourSeq 490 1 562 3000 93.8% chr7 - 35765402 36025294 259893 browser details YourSeq 490 1 562 3000 94.6% chr5 + 144637771 144638433 663 browser details YourSeq 489 1 562 3000 93.8% chr7 + 19351229 19908370 557142 browser details YourSeq 488 1 563 3000 94.4% chr7 + 30304342 30305116 775 browser details YourSeq 487 1 562 3000 94.3% chr5 - 134043386 134796960 753575 browser details YourSeq 487 1 561 3000 93.8% chr7 + 34922581 34923142 562 browser details YourSeq 486 1 570 3000 93.8% chr5 - 134043166 134043739 574 browser details YourSeq 485 4 562 3000 93.6% chr7 - 24375384 25111795 736412 browser details YourSeq 485 1 587 3000 92.3% chr5 + 141723545 141724230 686 Note: The 3000 bp section upstream of Exon 4 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 - 140903375 140906374 3000 browser details YourSeq 133 2803 3000 3000 93.0% chr12 + 77874268 77874534 267 browser details YourSeq 131 2808 3000 3000 91.9% chr12 + 108029238 108029529 292 browser details YourSeq 129 2809 3000 3000 92.2% chr12 + 109062536 109063003 468 browser details YourSeq 128 2803 2964 3000 95.8% chr11 + 93852302 93852510 209 browser details YourSeq 123 2818 2967 3000 93.7% chr11 + 51385185 51575050 189866 browser details YourSeq 117 2827 2999 3000 94.7% chr12 + 106428620 106428929 310 browser details YourSeq 117 2339 3000 3000 92.2% chr1 + 118822279 119039766 217488 browser details YourSeq 115 2801 2967 3000 90.8% chr14 - 31526640 31526822 183 browser details YourSeq 113 2801 2966 3000 93.9% chr11 + 51575119 51575303 185 browser details YourSeq 107 2818 2965 3000 83.6% chr14 + 111110413 111110548 136 browser details YourSeq 107 2805 2967 3000 84.0% chr10 + 79641293 79641447 155 browser details YourSeq 106 2806 3000 3000 88.7% chr3 - 144109140 144109326 187 browser details YourSeq 104 2808 2963 3000 94.1% chr12 + 109062731 109062986 256 browser details YourSeq 103 2803 2967 3000 85.9% chr11 - 119583216 119583344 129 browser details YourSeq 102 2827 2991 3000 86.4% chr10 + 79641231 79641383 153 browser details YourSeq 98 2807 2947 3000 93.9% chr5 - 112579626 112579797 172 browser details YourSeq 93 2803 2967 3000 81.5% chr13 - 103756200 103756328 129 browser details YourSeq 91 2829 2961 3000 97.0% chr15 + 28039161 28039458 298 browser details YourSeq 87 2803 2956 3000 94.8% chr16 - 11625332 11625560 229 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Card11 caspase recruitment domain family, member 11 [ Mus musculus (house mouse) ] Gene ID: 108723, updated on 24-Sep-2019 Gene summary Official Symbol Card11 provided by MGI Official Full Name caspase recruitment domain family, member 11 provided by MGI Primary source MGI:MGI:1916978 See related Ensembl:ENSMUSG00000036526 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 BIMP3; CARMA1; 0610008L17Rik; 2410011D02Rik Expression Biased expression in spleen adult (RPKM 26.7), thymus adult (RPKM 15.8) and 6 other tissuesS ee more Orthologs human all Genomic context Location: 5; 5 G2 See Card11 in Genome Data Viewer Exon count: 25 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (140872999..141000596, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (141348953..141476550, complement) Chromosome 5 - NC_000071.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Card11 ENSMUSG00000036526 Description caspase recruitment domain family, member 11 [Source:MGI Symbol;Acc:MGI:1916978] Gene Synonyms 0610008L17Rik, 2410011D02Rik, BIMP3, CARMA1 Location Chromosome 5: 140,872,990-141,000,582 reverse strand. GRCm38:CM000998.2 About this gene This gene has 3 transcripts (splice variants), 189 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 56 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Card11-201 ENSMUST00000085786.6 4107 1154aa ENSMUSP00000082941.5 Protein coding CCDS51686 Q8CIS0 TSL:1 GENCODE basic APPRIS P1 Card11-202 ENSMUST00000196169.1 1299 No protein - Retained intron - - TSL:1 Card11-203 ENSMUST00000199091.1 1067 No protein - Retained intron - - TSL:1 147.59 kb Forward strand 140.90Mb 140.95Mb 141.00Mb Contigs AC131725.2 > Genes (Comprehensive set... < Card11-201protein coding < Card11-202retained intron < Card11-203retained intron Regulatory Build 140.90Mb 140.95Mb 141.00Mb Reverse strand 147.59 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000085786 < Card11-201protein coding Reverse strand 127.59 kb ENSMUSP00000082... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Death-like domain superfamily PDZ superfamily P-loop containing nucleoside triphosphate hydrolase Pfam CARD domain PROSITE profiles CARD domain PANTHER PTHR14559 Caspase recruitment domain-containing protein 11 Gene3D 1.10.533.10 2.30.42.10 2.30.30.40 3.40.50.300 CDD CARD11, CARD domain cd00992 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1154 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.