https://www.alphaknockout.com Mouse Spock1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Spock1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Spock1 gene (NCBI Reference Sequence: NM_009262 ; Ensembl: ENSMUSG00000056222 ) is located on Mouse chromosome 13. 12 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 12 (Transcript: ENSMUST00000185502). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Spock1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-124L8 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 display no obvious morphological or behavioral abnormalities, are fertile, and have normal life spans. Adult homozygotes exhibit normal brain morphology and EEG recordings. Exon 5 starts from about 18.25% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 108376 bp, and the size of intron 5 for 3'-loxP site insertion: 30812 bp. The size of effective cKO region: ~615 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 gRNA region 5' gRNA region 3' 1 5 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Spock1 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(7115bp) | A(24.46% 1740) | C(23.63% 1681) | T(28.0% 1992) | G(23.92% 1702) 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 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% chr13 - 57587984 57590983 3000 browser details YourSeq 301 1062 1458 3000 90.2% chr14 - 87299221 87299618 398 browser details YourSeq 262 1062 1416 3000 91.0% chr18 - 60588571 60589212 642 browser details YourSeq 260 1068 1502 3000 91.5% chr14 + 18175620 18199144 23525 browser details YourSeq 258 1062 1508 3000 85.1% chr6 + 73516312 73516664 353 browser details YourSeq 256 1028 1501 3000 86.5% chr13 + 104866253 104866670 418 browser details YourSeq 255 1056 1502 3000 87.2% chr18 - 31210937 31211262 326 browser details YourSeq 252 1062 1501 3000 84.3% chr6 - 12631193 12631558 366 browser details YourSeq 252 1058 1506 3000 85.3% chr1 + 178230546 178230899 354 browser details YourSeq 251 1068 1497 3000 85.9% chr14 - 29848323 29848654 332 browser details YourSeq 251 1068 1501 3000 87.6% chr10 + 75983271 75983606 336 browser details YourSeq 247 1061 1502 3000 91.4% chr5 + 116873739 116874447 709 browser details YourSeq 244 1062 1500 3000 86.4% chr4 + 150880926 150881241 316 browser details YourSeq 243 1062 1501 3000 85.9% chr16 - 10799970 10800283 314 browser details YourSeq 240 1062 1502 3000 86.1% chr4 - 115083787 115084117 331 browser details YourSeq 240 1061 1503 3000 87.5% chr6 + 100405589 100405924 336 browser details YourSeq 240 1061 1502 3000 84.9% chr3 + 56243699 56244026 328 browser details YourSeq 239 1062 1501 3000 85.6% chrX - 12164066 12164412 347 browser details YourSeq 239 1062 1501 3000 85.8% chr2 - 119668108 119668448 341 browser details YourSeq 238 1062 1501 3000 85.5% chr1 + 33650299 33650644 346 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% chr13 - 57584369 57587368 3000 browser details YourSeq 67 783 912 3000 82.8% chr11 - 77099585 77099712 128 browser details YourSeq 62 807 913 3000 94.3% chr6 + 51306039 51306151 113 browser details YourSeq 58 775 876 3000 78.9% chr13 + 65090087 65090186 100 browser details YourSeq 55 783 872 3000 82.2% chrX + 102474441 102474527 87 browser details YourSeq 54 783 868 3000 79.3% chrX - 143727591 143727673 83 browser details YourSeq 53 806 871 3000 92.2% chr4 + 88968999 88969064 66 browser details YourSeq 52 819 913 3000 89.4% chr15 + 55586906 55587008 103 browser details YourSeq 52 785 859 3000 86.0% chr13 + 101067186 101067258 73 browser details YourSeq 52 806 875 3000 84.1% chr13 + 14635265 14635333 69 browser details YourSeq 51 785 870 3000 83.7% chr5 - 51847758 51847839 82 browser details YourSeq 51 819 911 3000 81.5% chr19 + 55686391 55686489 99 browser details YourSeq 49 806 874 3000 85.6% chr2 - 47951418 47951486 69 browser details YourSeq 49 806 870 3000 87.7% chr2 + 108677145 108677209 65 browser details YourSeq 45 806 870 3000 84.7% chr2 - 94763077 94763141 65 browser details YourSeq 45 786 846 3000 88.3% chr3 + 105778848 105778906 59 browser details YourSeq 44 771 977 3000 64.0% chr8 + 112421689 112421768 80 browser details YourSeq 43 808 866 3000 86.5% chr6 - 81254586 81254644 59 browser details YourSeq 43 810 879 3000 92.2% chr10 - 96476083 96476154 72 browser details YourSeq 43 809 869 3000 85.3% chr1 - 87738196 87738256 61 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: Spock1 sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 [ Mus musculus (house mouse) ] Gene ID: 20745, updated on 12-Aug-2019 Gene summary Official Symbol Spock1 provided by MGI Official Full Name sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 provided by MGI Primary source MGI:MGI:105371 See related Ensembl:ENSMUSG00000056222 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 Spock; Ticn1; testican Expression Biased expression in cerebellum adult (RPKM 21.7), cortex adult (RPKM 19.5) and 7 other tissuesS ee more Orthologs human all Genomic context Location: 13; 13 B1 See Spock1 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (57421195..57908332, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (57522556..58009693, complement) Chromosome 13 - NC_000079.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Spock1 ENSMUSG00000056222 Description sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 [Source:MGI Symbol;Acc:MGI:105371] Gene Synonyms Ticn1, testican 1 Location Chromosome 13: 57,421,195-57,908,332 reverse strand. GRCm38:CM001006.2 About this gene This gene has 6 transcripts (splice variants), 196 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Spock1-202 ENSMUST00000185502.6 4614 442aa ENSMUSP00000140409.1 Protein coding CCDS79195 Q6DIC9 TSL:5 GENCODE basic Spock1-206 ENSMUST00000189373.7 3128 367aa ENSMUSP00000139863.1 Protein coding CCDS79194 A0A087WPP6 TSL:5 GENCODE basic APPRIS P4 Spock1-205 ENSMUST00000187852.6 3113 364aa ENSMUSP00000141130.1 Protein coding CCDS79193 Q8BM07 TSL:1 GENCODE basic APPRIS ALT2 Spock1-204 ENSMUST00000186271.6 2005 439aa ENSMUSP00000140755.1 Protein coding CCDS49279 Q8BKQ3 TSL:1 GENCODE basic Spock1-201 ENSMUST00000172326.2 1320 439aa ENSMUSP00000128840.1 Protein coding CCDS49279 Q8BKQ3 TSL:5 GENCODE basic Spock1-203 ENSMUST00000185905.1 2794 343aa ENSMUSP00000153001.1 Protein coding - A0A286YCI6 TSL:1 GENCODE basic APPRIS ALT2 507.14 kb Forward strand 57.5Mb 57.6Mb 57.7Mb 57.8Mb 57.9Mb Contigs AC154748.2 > < AC154210.2 CT010428.13 > AC142258.4 > Genes (Comprehensive set... < Spock1-205protein coding < Spock1-206protein coding < Spock1-202protein coding < Spock1-204protein coding < Spock1-201protein coding < Spock1-203protein coding Regulatory Build 57.5Mb 57.6Mb 57.7Mb 57.8Mb 57.9Mb Reverse strand 507.14 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000185502 < Spock1-202protein coding Reverse strand 482.18 kb ENSMUSP00000140... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily EF-hand domain pair Kazal domain superfamily Thyroglobulin type-1 superfamily SMART Kazal domain Thyroglobulin type-1 Pfam Kazal domain Thyroglobulin type-1 SPARC/Testican, calcium-binding domain PROSITE profiles Kazal domain Thyroglobulin type-1 PROSITE patterns Thyroglobulin type-1 PANTHER PTHR13866 PTHR13866:SF17 Gene3D 3.30.60.30 1.10.238.10 Thyroglobulin type-1 superfamily CDD cd00104 Thyroglobulin type-1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 442 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.