https://www.alphaknockout.com Mouse Spa17 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Spa17 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Spa17 gene (NCBI Reference Sequence: NM_011449 ; Ensembl: ENSMUSG00000001948 ) is located on Mouse chromosome 9. 5 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000002013). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Spa17 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-443I14 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: Exon 3 starts from about 34.68% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 4148 bp, and the size of intron 4 for 3'-loxP site insertion: 2306 bp. The size of effective cKO region: ~2395 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 3 4 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Spa17 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(8895bp) | A(32.12% 2857) | C(19.28% 1715) | T(30.87% 2746) | G(17.73% 1577) 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% chr9 - 37607938 37610937 3000 browser details YourSeq 60 1219 1323 3000 88.4% chr2 + 132407771 132407877 107 browser details YourSeq 60 1219 1350 3000 95.6% chr2 + 27193416 27193621 206 browser details YourSeq 58 1201 1317 3000 86.5% chr5 + 125235706 125235821 116 browser details YourSeq 54 1221 1317 3000 89.8% chr2 + 167551126 167551231 106 browser details YourSeq 53 1230 1318 3000 88.6% chr7 - 127264895 127264981 87 browser details YourSeq 53 1228 1316 3000 85.8% chr3 - 69251120 69251206 87 browser details YourSeq 53 1219 1317 3000 88.9% chr13 + 103979155 103979252 98 browser details YourSeq 52 1174 1317 3000 96.5% chr1 + 79952567 79952747 181 browser details YourSeq 51 1219 1317 3000 91.3% chr5 - 66256400 66256497 98 browser details YourSeq 51 1216 1311 3000 82.8% chr12 - 16481419 16481504 86 browser details YourSeq 50 1222 1314 3000 91.1% chr1 - 135318755 135318846 92 browser details YourSeq 49 1273 1344 3000 93.0% chr11 - 6535998 6550642 14645 browser details YourSeq 49 1230 1312 3000 86.3% chr8 + 119556887 119556967 81 browser details YourSeq 48 1230 1317 3000 85.2% chr9 + 106055494 106055577 84 browser details YourSeq 48 1230 1315 3000 85.0% chr6 + 31506448 31506531 84 browser details YourSeq 47 1220 1317 3000 87.8% chr3 - 99321891 99321987 97 browser details YourSeq 46 1219 1317 3000 90.2% chr9 - 65066655 65066751 97 browser details YourSeq 46 1219 1297 3000 79.5% chr17 + 46881201 46881279 79 browser details YourSeq 44 1230 1318 3000 82.0% chr3 - 27696890 27696971 82 Note: The 3000 bp section upstream of Exon 3 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% chr9 - 37602543 37605542 3000 browser details YourSeq 130 1447 1623 3000 87.7% chr7 + 57180934 57181100 167 browser details YourSeq 130 1451 1611 3000 90.3% chr1 + 153647547 153647696 150 browser details YourSeq 130 1451 1599 3000 95.8% chr1 + 91247641 91247790 150 browser details YourSeq 129 1447 1577 3000 99.3% chr2 - 24106571 24106701 131 browser details YourSeq 129 1441 1577 3000 97.9% chr1 - 33773511 33773681 171 browser details YourSeq 127 1447 1577 3000 98.5% chr1 + 120451006 120451136 131 browser details YourSeq 123 1447 1577 3000 95.4% chrX - 84093230 84093359 130 browser details YourSeq 123 1449 1577 3000 97.7% chr6 - 38467179 38467307 129 browser details YourSeq 123 1452 1603 3000 88.7% chr2 - 13075011 13075154 144 browser details YourSeq 123 1451 1577 3000 98.5% chr1 - 180426920 180427046 127 browser details YourSeq 123 1451 1577 3000 98.5% chr16 + 55965830 55965956 127 browser details YourSeq 123 1451 1577 3000 98.5% chr12 + 4858392 4858518 127 browser details YourSeq 123 1447 1577 3000 97.0% chr11 + 78135327 78135457 131 browser details YourSeq 122 1447 1577 3000 94.6% chr14 + 120290633 120290761 129 browser details YourSeq 121 1454 1590 3000 94.9% chr7 - 120827407 120827704 298 browser details YourSeq 121 1451 1577 3000 97.7% chr7 + 118750251 118750377 127 browser details YourSeq 121 1451 1577 3000 97.7% chr7 + 113298627 113298753 127 browser details YourSeq 120 1452 1577 3000 97.7% chr9 - 64183275 64183400 126 browser details YourSeq 120 1416 1577 3000 93.5% chr15 - 10446054 10446617 564 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Spa17 sperm autoantigenic protein 17 [ Mus musculus (house mouse) ] Gene ID: 20686, updated on 12-Aug-2019 Gene summary Official Symbol Spa17 provided by MGI Official Full Name sperm autoantigenic protein 17 provided by MGI Primary source MGI:MGI:1333778 See related Ensembl:ENSMUSG00000001948 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 Sp17 Expression Biased expression in testis adult (RPKM 108.0) and CNS E11.5 (RPKM 4.0) See more Orthologs human all Genomic context Location: 9; 9 A4 See Spa17 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (37603294..37613916, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (37410883..37421207, complement) Chromosome 9 - NC_000075.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Spa17 ENSMUSG00000001948 Description sperm autoantigenic protein 17 [Source:MGI Symbol;Acc:MGI:1333778] Gene Synonyms Sp17, band 34 Location Chromosome 9: 37,603,290-37,613,722 reverse strand. GRCm38:CM001002.2 About this gene This gene has 3 transcripts (splice variants), 149 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Spa17- ENSMUST00000002013.10 696 149aa ENSMUSP00000002013.9 Protein coding CCDS22982 Q545M2 Q62252 TSL:1 201 GENCODE basic APPRIS P1 Spa17- ENSMUST00000214786.1 730 130aa ENSMUSP00000150651.1 Protein coding - A0A1L1SU87 CDS 3' incomplete 202 TSL:3 Spa17- ENSMUST00000215336.1 419 No - Retained intron - - TSL:2 203 protein 30.43 kb Forward strand 37.60Mb 37.61Mb 37.62Mb Genes Siae-202 >protein coding (Comprehensive set... Siae-206 >protein coding Siae-205 >retained intron Siae-201 >protein coding Siae-204 >protein coding Siae-207 >retained intron Contigs AC160125.2 > Genes (Comprehensive set... < Spa17-201protein coding < Spa17-202protein coding < Spa17-203retained intron Regulatory Build 37.60Mb 37.61Mb 37.62Mb Reverse strand 30.43 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000002013 < Spa17-201protein coding Reverse strand 10.43 kb ENSMUSP00000002... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF47391 SMART cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain IQ motif, EF-hand binding site Pfam cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain IQ motif, EF-hand binding site PROSITE profiles IQ motif, EF-hand binding site PIRSF Sperm surface protein Sp17 PANTHER Sperm surface protein Sp17 PTHR10699 Gene3D 1.20.890.10 1.20.5.190 CDD cd12100 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 149 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.