https://www.alphaknockout.com Mouse Cracr2a Knockout Project (CRISPR/Cas9) Objective: To create a Cracr2a knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cracr2a gene (NCBI Reference Sequence: NM_001033464 ; Ensembl: ENSMUSG00000061414 ) is located on Mouse chromosome 6. 7 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000071563). Exon 2~5 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 2 starts from the coding region. Exon 2~5 covers 70.22% of the coding region. The size of effective KO region: ~7703 bp. The KO region does not have any other known gene. 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 7 Legends Exon of mouse Cracr2a Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 5 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.9% 538) | C(23.35% 467) | T(28.65% 573) | G(21.1% 422) Note: The 2000 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.6% 512) | C(24.55% 491) | T(25.9% 518) | G(23.95% 479) Note: The 2000 bp section downstream of Exon 5 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr6 + 127601864 127603863 2000 browser details YourSeq 88 620 766 2000 84.5% chr2 - 14974852 14975000 149 browser details YourSeq 79 650 804 2000 87.1% chr1 - 84950927 84951087 161 browser details YourSeq 79 628 739 2000 85.8% chr18 + 77588725 77588838 114 browser details YourSeq 77 632 730 2000 88.9% chr2 - 49873757 49873855 99 browser details YourSeq 76 599 745 2000 88.8% chr2 + 143769808 143770341 534 browser details YourSeq 72 654 762 2000 83.5% chr16 + 20602434 20602544 111 browser details YourSeq 71 650 754 2000 79.8% chr8 + 78646303 78646396 94 browser details YourSeq 71 641 738 2000 86.8% chr5 + 126760736 126760839 104 browser details YourSeq 71 645 757 2000 79.5% chr1 + 39998210 39998321 112 browser details YourSeq 70 640 821 2000 92.7% chr7 + 126320063 126320460 398 browser details YourSeq 69 645 739 2000 86.4% chr10 + 45317484 45317578 95 browser details YourSeq 67 650 762 2000 87.7% chrX + 94307557 94307671 115 browser details YourSeq 66 611 739 2000 76.0% chr11 - 117145896 117146026 131 browser details YourSeq 65 627 717 2000 85.8% chr8 - 109753556 109753646 91 browser details YourSeq 65 611 721 2000 91.2% chr7 - 134076518 134076629 112 browser details YourSeq 65 646 727 2000 90.3% chr4 - 150726709 151140331 413623 browser details YourSeq 65 677 766 2000 86.7% chr16 - 21392238 21392328 91 browser details YourSeq 64 650 732 2000 89.5% chr16 + 97482519 97482600 82 browser details YourSeq 63 691 769 2000 91.2% chr4 - 47252484 47252844 361 Note: The 2000 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 2000 1 2000 2000 100.0% chr6 + 127611534 127613533 2000 browser details YourSeq 56 192 259 2000 95.3% chr13 - 38577304 38577427 124 browser details YourSeq 55 192 254 2000 95.3% chr9 + 22828178 22828252 75 browser details YourSeq 53 192 256 2000 91.4% chr2 + 58403078 58403140 63 browser details YourSeq 52 192 256 2000 93.6% chr10 - 78111195 78111299 105 browser details YourSeq 49 192 248 2000 94.5% chr8 - 113786086 113786144 59 browser details YourSeq 48 194 263 2000 81.2% chr2 - 76853292 76853347 56 browser details YourSeq 40 194 252 2000 95.6% chr9 + 45404683 45404741 59 browser details YourSeq 39 194 240 2000 93.2% chr8 - 81577706 81577762 57 browser details YourSeq 37 162 220 2000 97.5% chr14 - 116129159 116129300 142 browser details YourSeq 37 192 234 2000 95.2% chr11 - 103124937 103124979 43 browser details YourSeq 37 197 238 2000 95.3% chr19 + 51581807 51581858 52 browser details YourSeq 36 193 232 2000 97.5% chr5 + 117003509 117003552 44 browser details YourSeq 34 192 226 2000 100.0% chr5 - 146725720 146725878 159 browser details YourSeq 34 1647 1721 2000 97.3% chr11 - 41454570 41454764 195 browser details YourSeq 34 1434 1468 2000 100.0% chr7 + 98225198 98225243 46 browser details YourSeq 32 1432 1464 2000 100.0% chr2 - 8608855 8608888 34 browser details YourSeq 32 192 228 2000 94.6% chr1 - 185078762 185078810 49 browser details YourSeq 32 194 228 2000 97.1% chr6 + 139384475 139384514 40 browser details YourSeq 32 1434 1465 2000 100.0% chr10 + 113609817 113609848 32 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Cracr2a calcium release activated channel regulator 2A [ Mus musculus (house mouse) ] Gene ID: 381812, updated on 24-Oct-2019 Gene summary Official Symbol Cracr2a provided by MGI Official Full Name calcium release activated channel regulator 2A provided by MGI Primary source MGI:MGI:2685919 See related Ensembl:ENSMUSG00000061414 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 Gm462; Gm1073; Efcab4b Expression Broad expression in colon adult (RPKM 2.8), lung adult (RPKM 2.7) and 16 other tissues See more Orthologs human all Genomic context Location: 6; 6 F3 See Cracr2a in Genome Data Viewer Exon count: 25 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (127561062..127674234) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (127527993..127579956) Chromosome 6 - NC_000072.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Cracr2a ENSMUSG00000061414 Description calcium release activated channel regulator 2A [Source:MGI Symbol;Acc:MGI:2685919] Gene Synonyms Efcab4b, LOC243645, LOC381812 Location Chromosome 6: 127,561,338-127,674,248 forward strand. GRCm38:CM000999.2 About this gene This gene has 3 transcripts (splice variants), 248 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cracr2a-201 ENSMUST00000071563.4 1094 310aa ENSMUSP00000071494.4 Protein coding CCDS20566 Q3UP38 TSL:1 GENCODE basic Cracr2a-203 ENSMUST00000212051.1 7625 726aa ENSMUSP00000148569.1 Protein coding - A0A1D5RLZ8 TSL:5 GENCODE basic APPRIS P1 Cracr2a-202 ENSMUST00000201303.4 1267 No protein - Retained intron - - TSL:1 132.91 kb Forward strand 127.56Mb 127.58Mb 127.60Mb 127.62Mb 127.64Mb 127.66Mb 127.68Mb Genes (Comprehensive set... Cracr2a-203 >protein coding Cracr2a-202 >retained intron Gm43634-201 >lncRNA Cracr2a-201 >protein coding Contigs AC173480.1 > AC127373.3 > Regulatory Build 127.56Mb 127.58Mb 127.60Mb 127.62Mb 127.64Mb 127.66Mb 127.68Mb Reverse strand 132.91 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000071563 51.96 kb Forward strand Cracr2a-201 >protein coding ENSMUSP00000071... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily EF-hand domain pair SMART EF-hand domain Pfam EF-hand domain PROSITE profiles EF-hand domain PROSITE patterns EF-Hand 1, calcium-binding site PANTHER PTHR22621 PTHR22621:SF27 Gene3D 1.10.238.10 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 310 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.