https://www.alphaknockout.com Mouse Srpx2 Knockout Project (CRISPR/Cas9) Objective: To create a Srpx2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Srpx2 gene (NCBI Reference Sequence: NM_001083895 ; Ensembl: ENSMUSG00000031253 ) is located on Mouse chromosome X. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000113304). Exon 4~10 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 null allele exhibit abnormal vocalization and response to social novelty. Exon 4 starts from about 12.32% of the coding region. Exon 4~10 covers 75.07% of the coding region. The size of effective KO region: ~6663 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 4 5 6 7 8 9 10 11 Legends Exon of mouse Srpx2 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 4 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 987 bp section downstream of Exon 10 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(25.25% 505) | C(20.1% 402) | T(38.4% 768) | G(16.25% 325) Note: The 2000 bp section upstream of Exon 4 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(987bp) | A(31.51% 311) | C(20.36% 201) | T(23.91% 236) | G(24.21% 239) Note: The 987 bp section downstream of Exon 10 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% chrX + 133921637 133923636 2000 browser details YourSeq 80 1692 1868 2000 75.8% chr1 + 50700011 50700178 168 browser details YourSeq 65 1740 1869 2000 80.3% chr14 + 56092531 56092653 123 browser details YourSeq 60 1747 1865 2000 79.3% chr13 + 69115614 69115724 111 browser details YourSeq 55 1623 1678 2000 100.0% chr19 - 30821706 30821773 68 browser details YourSeq 51 1686 1759 2000 91.9% chr7 + 50658359 50658435 77 browser details YourSeq 48 1807 1869 2000 89.3% chr8 - 103379529 103379590 62 browser details YourSeq 48 1715 1850 2000 90.0% chr8 - 38565780 38565943 164 browser details YourSeq 48 1806 1869 2000 88.0% chr1 - 62232700 62232762 63 browser details YourSeq 48 1806 1867 2000 88.0% chr12 + 71467730 71467790 61 browser details YourSeq 46 1672 1844 2000 94.3% chrX - 165578698 165578874 177 browser details YourSeq 46 1810 1867 2000 90.4% chr9 - 92510471 92510527 57 browser details YourSeq 46 1623 1854 2000 66.1% chr10 + 88864424 88864542 119 browser details YourSeq 45 1101 1175 2000 76.0% chr5 - 24910925 24910984 60 browser details YourSeq 45 1665 1869 2000 84.7% chr14 + 21539120 21539320 201 browser details YourSeq 45 1633 1841 2000 92.5% chr1 + 16835664 16835877 214 browser details YourSeq 44 1806 1868 2000 94.0% chr15 - 93023060 93023160 101 browser details YourSeq 43 1806 1868 2000 86.8% chr1 + 137385288 137385349 62 browser details YourSeq 41 1673 1841 2000 90.2% chr6 + 24798756 24798925 170 browser details YourSeq 40 1690 1825 2000 64.8% chr11 - 12212874 12213009 136 Note: The 2000 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 987 1 987 987 100.0% chrX + 133930300 133931286 987 browser details YourSeq 24 891 920 987 76.0% chr11 - 114368114 114368138 25 browser details YourSeq 20 820 839 987 100.0% chr1 - 172238908 172238927 20 Note: The 987 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Srpx2 sushi-repeat-containing protein, X-linked 2 [ Mus musculus (house mouse) ] Gene ID: 68792, updated on 12-Aug-2019 Gene summary Official Symbol Srpx2 provided by MGI Official Full Name sushi-repeat-containing protein, X-linked 2 provided by MGI Primary source MGI:MGI:1916042 See related Ensembl:ENSMUSG00000031253 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 SRP; SRPUL; 1110039C07Rik Expression Broad expression in subcutaneous fat pad adult (RPKM 8.1), mammary gland adult (RPKM 5.6) and 18 other tissues See Orthologs more human all Genomic context Location: X; X E3 See Srpx2 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (133908416..133932448) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (130442965..130467001) Chromosome X - NC_000086.7 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Srpx2 ENSMUSG00000031253 Description sushi-repeat-containing protein, X-linked 2 [Source:MGI Symbol;Acc:MGI:1916042] Gene Synonyms 1110039C07Rik Location Chromosome X: 133,908,426-133,932,446 forward strand. GRCm38:CM001013.2 About this gene This gene has 2 transcripts (splice variants), 192 orthologues, 38 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Srpx2-202 ENSMUST00000113304.1 2848 468aa ENSMUSP00000108929.2 Protein coding CCDS41119 Q8R054 TSL:1 GENCODE basic APPRIS P1 Srpx2-201 ENSMUST00000033606.14 2290 468aa ENSMUSP00000033606.9 Protein coding CCDS41119 Q8R054 TSL:1 GENCODE basic APPRIS P1 44.02 kb Forward strand 133.90Mb 133.91Mb 133.92Mb 133.93Mb 133.94Mb Genes (Comprehensive set... Srpx2-201 >protein coding Srpx2-202 >protein coding Contigs AL691421.9 > Genes < Tspan6-201protein coding < Sytl4-201protein coding (Comprehensive set... < 4930570D08Rik-201lncRNA < Sytl4-203protein coding Regulatory Build 133.90Mb 133.91Mb 133.92Mb 133.93Mb 133.94Mb Reverse strand 44.02 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000113304 24.00 kb Forward strand Srpx2-202 >protein coding ENSMUSP00000108... Low complexity (Seg) Cleavage site (Sign... Superfamily Sushi/SCR/CCP superfamily SMART Sushi/SCR/CCP domain Pfam Sushi/SCR/CCP domain Domain of unknown function DUF4174 HYR domain PROSITE profiles Sushi/SCR/CCP domain HYR domain PANTHER PTHR46343 Sushi repeat-containing protein SRPX2 Gene3D 2.10.70.10 CDD Sushi/SCR/CCP domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 468 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.