https://www.alphaknockout.com Mouse Asz1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Asz1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Asz1 gene (NCBI Reference Sequence: NM_023729 ; Ensembl: ENSMUSG00000010796 ) is located on Mouse chromosome 6. 13 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 13 (Transcript: ENSMUST00000010940). 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 Asz1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-39F14 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: Homozygous null male mice are sterile resulting from a block in spermatid development. Exon 3 starts from about 14.46% 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: 3504 bp, and the size of intron 4 for 3'-loxP site insertion: 26235 bp. The size of effective cKO region: ~1899 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 13 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Asz1 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(8399bp) | A(30.94% 2599) | C(17.18% 1443) | T(34.27% 2878) | G(17.61% 1479) 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% chr6 - 18105149 18108148 3000 browser details YourSeq 90 2066 2231 3000 79.5% chr15 - 85514725 85514858 134 browser details YourSeq 85 2052 2231 3000 83.9% chr1 + 160949692 160949863 172 browser details YourSeq 81 1684 2231 3000 88.6% chr17 - 33656370 33656931 562 browser details YourSeq 73 1598 2232 3000 71.2% chr13 + 49291520 49291692 173 browser details YourSeq 71 2141 2231 3000 85.3% chr17 + 26862363 26862450 88 browser details YourSeq 71 2092 2226 3000 83.2% chr10 + 25084311 25084450 140 browser details YourSeq 70 2141 2251 3000 83.0% chr18 + 32935555 32935663 109 browser details YourSeq 69 2057 2226 3000 91.5% chr2 + 146057875 146058216 342 browser details YourSeq 68 2096 2198 3000 91.7% chr8 + 105740876 105740982 107 browser details YourSeq 66 2076 2226 3000 79.8% chr18 - 67328356 67328479 124 browser details YourSeq 66 2140 2255 3000 92.5% chr2 + 158828183 158828623 441 browser details YourSeq 66 2141 2231 3000 89.2% chr15 + 89742405 89742492 88 browser details YourSeq 66 2084 2231 3000 92.4% chr1 + 182381490 182381641 152 browser details YourSeq 65 1973 2216 3000 95.8% chr9 - 120762182 120762560 379 browser details YourSeq 64 2143 2226 3000 89.1% chr1 + 63135226 63135306 81 browser details YourSeq 63 2141 2231 3000 83.4% chr4 - 57912895 57912981 87 browser details YourSeq 62 2144 2251 3000 81.4% chr13 - 100605571 100605675 105 browser details YourSeq 62 2141 2231 3000 84.0% chr8 + 25183403 25183490 88 browser details YourSeq 62 2140 2225 3000 82.0% chr5 + 33106917 33106999 83 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% chr6 - 18100250 18103249 3000 browser details YourSeq 376 1783 2188 3000 96.4% chr18 - 61465282 61465687 406 browser details YourSeq 372 1783 2188 3000 95.1% chr6 + 142092773 142093175 403 browser details YourSeq 371 1783 2188 3000 95.9% chr12 + 12036297 12046040 9744 browser details YourSeq 370 1783 2188 3000 95.6% chr5 + 72418286 72418691 406 browser details YourSeq 368 1779 2188 3000 95.6% chr3 - 36118021 36118434 414 browser details YourSeq 367 1725 2188 3000 92.6% chr18 - 84829653 84830083 431 browser details YourSeq 367 1784 2188 3000 94.6% chr16 - 64570902 64571303 402 browser details YourSeq 367 1783 2188 3000 94.9% chr12 + 119086921 119087325 405 browser details YourSeq 366 1782 2188 3000 95.8% chr6 - 43120152 43120559 408 browser details YourSeq 366 1783 2188 3000 94.4% chr1 - 113566498 113566901 404 browser details YourSeq 366 1783 2188 3000 94.1% chr12 + 78180439 78180840 402 browser details YourSeq 365 1783 2188 3000 95.6% chr1 + 157926983 157927390 408 browser details YourSeq 364 1783 2188 3000 94.1% chrX - 10538377 10538779 403 browser details YourSeq 364 1783 2188 3000 94.1% chr7 - 55291311 55291713 403 browser details YourSeq 363 1784 2188 3000 94.4% chrX - 23546172 23546575 404 browser details YourSeq 363 1657 2175 3000 91.7% chr12 - 57343520 57343956 437 browser details YourSeq 362 1783 2188 3000 93.8% chrX - 98521644 98522046 403 browser details YourSeq 361 1783 2188 3000 94.0% chr9 - 15124971 15125372 402 browser details YourSeq 361 1785 2188 3000 94.8% chr6 + 50648852 50649256 405 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: Asz1 ankyrin repeat, SAM and basic leucine zipper domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 74068, updated on 12-Aug-2019 Gene summary Official Symbol Asz1 provided by MGI Official Full Name ankyrin repeat, SAM and basic leucine zipper domain containing 1 provided by MGI Primary source MGI:MGI:1921318 See related Ensembl:ENSMUSG00000010796 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 Gasz; ORF3; 4933400N19Rik Expression Biased expression in bladder adult (RPKM 3.4), testis adult (RPKM 3.0) and 1 other tissue See more Orthologs human all Genomic context Location: 6; 6 A2 See Asz1 in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (18050964..18109061, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (18000964..18059061, complement) Chromosome 6 - NC_000072.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Asz1 ENSMUSG00000010796 Description ankyrin repeat, SAM and basic leucine zipper domain containing 1 [Source:MGI Symbol;Acc:MGI:1921318] Gene Synonyms 4933400N19Rik, Gasz, ORF3 Location Chromosome 6: 18,050,964-18,109,061 reverse strand. GRCm38:CM000999.2 About this gene This gene has 5 transcripts (splice variants), 133 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Asz1- ENSMUST00000010940.10 1736 475aa ENSMUSP00000010940.4 Protein coding CCDS19929 Q8VD46 TSL:1 201 GENCODE basic APPRIS P1 Asz1- ENSMUST00000136194.1 694 148aa ENSMUSP00000122680.1 Nonsense mediated - D6RFV1 TSL:5 203 decay Asz1- ENSMUST00000136626.7 2345 No - Retained intron - - TSL:5 204 protein Asz1- ENSMUST00000146216.1 297 No - Retained intron - - TSL:5 205 protein Asz1- ENSMUST00000131614.1 667 No - lncRNA - - TSL:2 202 protein 78.10 kb Forward strand 18.06Mb 18.08Mb 18.10Mb Contigs AC068561.2 > AC158647.2 > Genes (Comprehensive set... < Asz1-201protein coding < Gm43845-201TEC < Asz1-204retained intron < Asz1-205retained intron < Asz1-203nonsense mediated decay < Asz1-202lncRNA Regulatory Build 18.06Mb 18.08Mb 18.10Mb Reverse strand 78.10 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000010940 < Asz1-201protein coding Reverse strand 58.09 kb ENSMUSP00000010... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Ankyrin repeat-containing domain superfamily Sterile alpha motif/pointed domain superfamily SSF140860 SMART Ankyrin repeat Sterile alpha motif domain Prints Ankyrin repeat Pfam PF13637 Ankyrin repeat-containing domain Sterile alpha motif domain PROSITE profiles Ankyrin repeat-containing domain Ankyrin repeat PANTHER PTHR24157 PTHR24157:SF3 Gene3D Ankyrin repeat-containing domain superfamily Sterile alpha motif/pointed domain superfamily CDD cd09521 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 475 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.