https://www.alphaknockout.com Mouse Tspyl5 Knockout Project (CRISPR/Cas9) Objective: To create a Tspyl5 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tspyl5 gene (NCBI Reference Sequence: NM_001085421 ; Ensembl: ENSMUSG00000038984 ) is located on Mouse chromosome 15. 1 exon is identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000042021). Exon 1 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 1 starts from about 0.08% of the coding region. Exon 1 covers 100.0% of the coding region. The size of effective KO region: ~1216 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 Legends Exon of mouse Tspyl5 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 start codon 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 2000 bp section downstream of stop codon 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(28.35% 567) | C(21.6% 432) | T(26.1% 522) | G(23.95% 479) Note: The 2000 bp section upstream of start codon 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(24.75% 495) | C(22.05% 441) | T(31.9% 638) | G(21.3% 426) Note: The 2000 bp section downstream of stop codon 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% chr15 - 33687798 33689797 2000 browser details YourSeq 436 5 594 2000 89.1% chr9 + 120682887 120683484 598 browser details YourSeq 417 5 611 2000 88.9% chr14 + 30688026 30688649 624 browser details YourSeq 415 5 608 2000 87.3% chr7 - 57637052 57637656 605 browser details YourSeq 414 5 598 2000 89.3% chr13 + 95297900 95298526 627 browser details YourSeq 413 5 607 2000 88.5% chr5 - 9366354 9366959 606 browser details YourSeq 413 5 594 2000 87.9% chr9 + 74983072 74983666 595 browser details YourSeq 412 19 594 2000 88.8% chr4 - 103044134 103044719 586 browser details YourSeq 411 5 612 2000 85.9% chr14 - 21916090 21916693 604 browser details YourSeq 411 29 611 2000 89.3% chr18 + 79908508 79909131 624 browser details YourSeq 411 19 606 2000 87.6% chr14 + 73439001 73439653 653 browser details YourSeq 409 25 605 2000 87.9% chr2 + 147505938 147506552 615 browser details YourSeq 408 5 592 2000 88.6% chr9 + 48312788 48313386 599 browser details YourSeq 407 5 606 2000 88.0% chr19 - 18034560 18035151 592 browser details YourSeq 405 28 594 2000 87.9% chr13 + 103925193 103925768 576 browser details YourSeq 404 5 606 2000 86.4% chr16 - 13277995 13278591 597 browser details YourSeq 403 25 589 2000 88.2% chr9 - 57783427 57783993 567 browser details YourSeq 402 5 594 2000 89.8% chr1 - 82609496 82610090 595 browser details YourSeq 400 10 610 2000 87.6% chr17 - 16470857 16471464 608 browser details YourSeq 400 5 591 2000 88.5% chr9 + 80082483 80083070 588 Note: The 2000 bp section upstream of start codon 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% chr15 - 33684580 33686579 2000 browser details YourSeq 48 1595 1929 2000 96.3% chr10 - 39638764 39918341 279578 browser details YourSeq 29 1311 1367 2000 94.2% chr12 - 58312434 58312492 59 browser details YourSeq 26 821 847 2000 100.0% chr1 + 15301896 15301939 44 browser details YourSeq 23 399 421 2000 100.0% chrY - 4290334 4290356 23 browser details YourSeq 23 1652 1674 2000 100.0% chr12 + 113241316 113241338 23 browser details YourSeq 21 1229 1249 2000 100.0% chr16 + 49798636 49798656 21 browser details YourSeq 20 1213 1238 2000 88.5% chr4 + 72859671 72859696 26 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Tspyl5 testis-specific protein, Y-encoded-like 5 [ Mus musculus (house mouse) ] Gene ID: 239364, updated on 12-Aug-2019 Gene summary Official Symbol Tspyl5 provided by MGI Official Full Name testis-specific protein, Y-encoded-like 5 provided by MGI Primary source MGI:MGI:2442458 See related Ensembl:ENSMUSG00000038984 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 6330530B20; E130308C19Rik Orthologs human all Genomic context Location: 15; 15 B3.1 See Tspyl5 in Genome Data Viewer Exon count: 1 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (33683875..33687883, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (33613630..33617638, complement) Chromosome 15 - NC_000081.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Tspyl5 ENSMUSG00000038984 Description testis-specific protein, Y-encoded-like 5 [Source:MGI Symbol;Acc:MGI:2442458] Gene Synonyms E130308C19Rik Location Chromosome 15: 33,683,875-33,687,884 reverse strand. GRCm38:CM001008.2 About this gene This gene has 1 transcript (splice variant), 183 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tspyl5-201 ENSMUST00000042021.4 4010 406aa ENSMUSP00000045542.3 Protein coding CCDS49591 Q69ZB3 TSL:NA GENCODE basic APPRIS P1 24.01 kb Forward strand 33.675Mb 33.680Mb 33.685Mb 33.690Mb 33.695Mb Contigs < AC129208.4 Genes (Comprehensive set... < Tspyl5-201protein coding Regulatory Build 33.675Mb 33.680Mb 33.685Mb 33.690Mb 33.695Mb Reverse strand 24.01 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000042021 < Tspyl5-201protein coding Reverse strand 4.01 kb ENSMUSP00000045... MobiDB lite Low complexity (Seg) Superfamily NAP-like superfamily Pfam Nucleosome assembly protein (NAP) PANTHER Nucleosome assembly protein (NAP) PTHR11875:SF30 Gene3D 3.30.1120.90 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 406 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.