https://www.alphaknockout.com Mouse Ss18l1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ss18l1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ss18l1 gene (NCBI Reference Sequence: NM_178750 ; Ensembl: ENSMUSG00000039086 ) is located on Mouse chromosome 2. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 11 (Transcript: ENSMUST00000041126). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ss18l1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-343C13 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 disruptions in this gene have central nervous system and coordination defects. They grow slowly and usually die before adulthood. Those that do survive are infertile. Exon 2 starts from about 5.8% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 9595 bp, and the size of intron 2 for 3'-loxP site insertion: 768 bp. The size of effective cKO region: ~577 bp. The cKO 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 2 3 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ss18l1 Homology arm cKO region loxP site Page 2 of 8 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(7077bp) | A(22.13% 1566) | C(25.7% 1819) | T(26.49% 1875) | G(25.67% 1817) 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 8 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% chr2 + 180049215 180052214 3000 browser details YourSeq 324 156 788 3000 92.6% chr14 + 34504894 34650926 146033 browser details YourSeq 270 132 724 3000 86.5% chr18 - 56692898 56693529 632 browser details YourSeq 213 165 721 3000 84.1% chr2 - 167003393 167003861 469 browser details YourSeq 203 465 744 3000 89.9% chr4 - 116129064 116355798 226735 browser details YourSeq 174 171 721 3000 81.8% chr7 - 119909254 119909577 324 browser details YourSeq 171 301 726 3000 83.3% chr15 + 25798299 25798622 324 browser details YourSeq 161 301 693 3000 91.3% chr9 - 56974354 56974963 610 browser details YourSeq 160 1406 1583 3000 95.6% chr1 + 33682642 33682845 204 browser details YourSeq 158 1333 1589 3000 90.0% chr7 - 97773456 97774001 546 browser details YourSeq 155 161 536 3000 86.8% chrX - 94594609 94594941 333 browser details YourSeq 147 1427 1594 3000 92.7% chr18 + 38986558 38986723 166 browser details YourSeq 147 479 722 3000 94.1% chr11 + 101249620 101250251 632 browser details YourSeq 144 1426 1589 3000 94.0% chr7 + 28632147 28632310 164 browser details YourSeq 142 1424 1588 3000 94.0% chr16 - 38309651 38309818 168 browser details YourSeq 141 465 693 3000 92.2% chr2 + 71157525 71665728 508204 browser details YourSeq 140 463 721 3000 92.7% chr3 + 88915271 88915536 266 browser details YourSeq 139 355 721 3000 83.6% chr6 - 87367283 87367500 218 browser details YourSeq 139 1253 1594 3000 84.8% chr17 + 66000180 66000502 323 browser details YourSeq 139 1423 1586 3000 91.4% chr11 + 106616456 106616618 163 Note: The 3000 bp section upstream of Exon 2 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% chr2 + 180052792 180055791 3000 browser details YourSeq 141 912 1280 3000 82.0% chr2 - 118390971 118391155 185 browser details YourSeq 133 1105 1282 3000 85.6% chr9 - 45837882 45838040 159 browser details YourSeq 133 1133 1290 3000 92.4% chr8 + 71411089 71411253 165 browser details YourSeq 133 1136 1295 3000 91.9% chr7 + 28964916 28965076 161 browser details YourSeq 133 1132 1282 3000 94.1% chr4 + 133564904 133565054 151 browser details YourSeq 132 1132 1294 3000 90.8% chr7 + 75829605 75829812 208 browser details YourSeq 132 1119 1282 3000 88.2% chr1 + 136585504 136585664 161 browser details YourSeq 131 1140 1292 3000 93.5% chr9 - 45374315 45374472 158 browser details YourSeq 131 1134 1296 3000 90.7% chr1 - 87439666 87439832 167 browser details YourSeq 131 1136 1289 3000 92.9% chr1 + 84673203 84673366 164 browser details YourSeq 130 1133 1280 3000 94.0% chr5 - 123774701 123774848 148 browser details YourSeq 130 1130 1290 3000 90.7% chr17 + 29760366 29760537 172 browser details YourSeq 130 1121 1279 3000 91.9% chr12 + 102875096 102875271 176 browser details YourSeq 130 1133 1282 3000 93.4% chr1 + 69867549 69867698 150 browser details YourSeq 129 1132 1286 3000 90.9% chr6 - 125263819 125263972 154 browser details YourSeq 128 1136 1288 3000 92.2% chr4 + 15919387 15919546 160 browser details YourSeq 128 1120 1282 3000 90.0% chr1 + 128411444 128411608 165 browser details YourSeq 128 1133 1282 3000 92.7% chr1 + 52019330 52019479 150 browser details YourSeq 127 1140 1294 3000 90.2% chr8 - 122801409 122801562 154 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Ss18l1 SS18, nBAF chromatin remodeling complex subunit like 1 [ Mus musculus (house mouse) ] Gene ID: 269397, updated on 14-Oct-2019 Gene summary Official Symbol Ss18l1 provided by MGI Official Full Name SS18, nBAF chromatin remodeling complex subunit like 1 provided by MGI Primary source MGI:MGI:2444061 See related Ensembl:ENSMUSG00000039086 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 CREST; A230053O16Rik Expression Ubiquitous expression in genital fat pad adult (RPKM 17.2), whole brain E14.5 (RPKM 15.9) and 26 other tissues See more Orthologs human all Genomic context Location: 2; 2 H4 See Ss18l1 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (180042483..180070201) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (179777188..179804906) Chromosome 2 - NC_000068.7 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Ss18l1 ENSMUSG00000039086 Description SS18, nBAF chromatin remodeling complex subunit like 1 [Source:MGI Symbol;Acc:MGI:2444061] Gene Synonyms A230053O16Rik, CREST Location Chromosome 2: 180,042,509-180,070,201 forward strand. GRCm38:CM000995.2 About this gene This gene has 1 transcript (splice variant), 195 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ss18l1-201 ENSMUST00000041126.8 4343 402aa ENSMUSP00000041288.8 Protein coding CCDS17167 Q8BW22 TSL:1 GENCODE basic APPRIS P1 Page 6 of 8 https://www.alphaknockout.com 47.69 kb Forward strand 180.04Mb 180.05Mb 180.06Mb 180.07Mb 180.08Mb Genes (Comprehensive set... Lsm14b-201 >protein coding Ss18l1-201 >protein coding Mtg2-206 >retained intron Lsm14b-202 >protein coding Mtg2-207 >nonsense mediated decay Lsm14b-203 >lncRNA Mtg2-204 >retained intron Mtg2-202 >protein coding Mtg2-203 >retained intron Mtg2-201 >protein coding Contigs AL663067.14 > AL672130.14 > Genes < Psma7-201protein coding (Comprehensive set... < Psma7-203protein coding < Psma7-202retained intron < Psma7-205retained intron < Psma7-206retained intron < Psma7-204retained intron < Psma7-207lncRNA Regulatory Build 180.04Mb 180.05Mb 180.06Mb 180.07Mb 180.08Mb Reverse strand 47.69 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000041126 27.69 kb Forward strand Ss18l1-201 >protein coding ENSMUSP00000041... MobiDB lite Low complexity (Seg) Pfam SS18 family PANTHER PTHR23107 PTHR23107:SF2 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 402 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.