https://www.alphaknockout.com Mouse Spock1 Knockout Project (CRISPR/Cas9) Objective: To create a Spock1 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 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: 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. Exon 5 covers 8.67% of the coding region. The size of effective KO region: ~115 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 gRNA region 5' gRNA region 3' 1 5 12 Legends Exon of mouse Spock1 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 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. 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. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. 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(24.1% 482) | C(25.85% 517) | T(27.15% 543) | G(22.9% 458) Note: The 2000 bp section upstream 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.5% 490) | C(22.1% 442) | T(30.05% 601) | G(23.35% 467) 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% chr13 - 57587734 57589733 2000 browser details YourSeq 191 1 251 2000 87.2% chr8 + 117910717 117910966 250 browser details YourSeq 183 4 253 2000 85.9% chr14 - 87299178 87299425 248 browser details YourSeq 172 2 241 2000 88.9% chr8 - 121268893 121269131 239 browser details YourSeq 170 1 251 2000 85.3% chr4 + 148188737 148188947 211 browser details YourSeq 166 13 219 2000 91.1% chr3 - 54923243 54923489 247 browser details YourSeq 165 1 219 2000 87.7% chr5 + 115824515 115824733 219 browser details YourSeq 164 4 251 2000 84.5% chr5 - 116898715 116898961 247 browser details YourSeq 160 1 205 2000 89.7% chr16 + 94809326 94809651 326 browser details YourSeq 158 6 206 2000 89.6% chr4 - 140378058 140564808 186751 browser details YourSeq 154 2 251 2000 84.1% chr1 - 23537002 23537213 212 browser details YourSeq 152 8 217 2000 92.8% chr4 - 131450380 131450768 389 browser details YourSeq 148 1 253 2000 84.5% chr8 - 119781588 119781818 231 browser details YourSeq 145 1 251 2000 80.0% chr13 + 38848425 38848635 211 browser details YourSeq 144 1 184 2000 89.2% chr5 - 74051046 74051229 184 browser details YourSeq 140 17 219 2000 93.3% chr18 - 75568156 75720448 152293 browser details YourSeq 138 8 261 2000 84.3% chr1 - 39100612 39100867 256 browser details YourSeq 133 1 252 2000 93.0% chr17 - 31903042 31903470 429 browser details YourSeq 131 13 251 2000 79.6% chr13 - 57480506 57480724 219 browser details YourSeq 131 1 219 2000 90.3% chr1 - 40947266 40947504 239 Note: The 2000 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 2000 1 2000 2000 100.0% chr13 - 57585619 57587618 2000 browser details YourSeq 67 1033 1162 2000 82.8% chr11 - 77099585 77099712 128 browser details YourSeq 62 240 344 2000 88.0% chrX + 12113325 12113476 152 browser details YourSeq 62 1057 1163 2000 94.3% chr6 + 51306039 51306151 113 browser details YourSeq 61 194 351 2000 91.7% chr11 - 51708280 51708478 199 browser details YourSeq 58 1025 1126 2000 78.9% chr13 + 65090087 65090186 100 browser details YourSeq 56 232 349 2000 88.9% chr15 - 37731740 37731895 156 browser details YourSeq 55 1033 1122 2000 82.2% chrX + 102474441 102474527 87 browser details YourSeq 54 1033 1118 2000 79.3% chrX - 143727591 143727673 83 browser details YourSeq 53 1056 1121 2000 92.2% chr4 + 88968999 88969064 66 browser details YourSeq 52 1069 1163 2000 89.4% chr15 + 55586906 55587008 103 browser details YourSeq 52 1035 1109 2000 86.0% chr13 + 101067186 101067258 73 browser details YourSeq 52 1056 1125 2000 84.1% chr13 + 14635265 14635333 69 browser details YourSeq 51 1035 1120 2000 83.7% chr5 - 51847758 51847839 82 browser details YourSeq 51 1069 1161 2000 81.5% chr19 + 55686391 55686489 99 browser details YourSeq 50 241 350 2000 91.9% chr17 + 56361966 56362077 112 browser details YourSeq 49 1056 1124 2000 85.6% chr2 - 47951418 47951486 69 browser details YourSeq 49 1056 1120 2000 87.7% chr2 + 108677145 108677209 65 browser details YourSeq 47 246 344 2000 94.5% chr13 + 104672622 104672789 168 browser details YourSeq 47 242 349 2000 89.9% chr1 + 84926223 84926387 165 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: 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 6 of 8 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 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000185502 < Spock1-202protein coding Reverse strand 482.18 kb ENSMUSP00000140... MobiDB lite Low