https://www.alphaknockout.com Mouse Zmiz1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Zmiz1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Zmiz1 gene (NCBI Reference Sequence: NM_183208 ; Ensembl: ENSMUSG00000007817 ) is located on Mouse chromosome 14. 24 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 24 (Transcript: ENSMUST00000162645). Exon 7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Zmiz1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-259E22 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 a null mutation display embryonic lethality during organogenesis with failure of yolk sac vascular remodeling and abnormal embryonic vascular development. Exon 7 starts from about 8.83% of the coding region. The knockout of Exon 7 will result in frameshift of the gene. The size of intron 6 for 5'-loxP site insertion: 54199 bp, and the size of intron 7 for 3'-loxP site insertion: 7540 bp. The size of effective cKO region: ~645 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 7 24 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Zmiz1 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. 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(7145bp) | A(23.12% 1652) | C(25.92% 1852) | T(25.68% 1835) | G(25.28% 1806) 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% chr14 + 25632624 25635623 3000 browser details YourSeq 166 2143 2377 3000 92.0% chr5 + 28549975 28550214 240 browser details YourSeq 166 2133 2367 3000 93.8% chr4 + 119152193 119152595 403 browser details YourSeq 163 2138 2376 3000 90.3% chr4 + 81103528 81103775 248 browser details YourSeq 161 2175 2376 3000 92.4% chrX - 129423121 129423352 232 browser details YourSeq 158 2143 2378 3000 90.4% chr3 + 23868053 23868315 263 browser details YourSeq 156 2176 2377 3000 91.7% chr6 + 70867644 70867876 233 browser details YourSeq 154 2175 2404 3000 89.4% chr10 + 55506593 55506843 251 browser details YourSeq 153 2173 2576 3000 81.8% chrX - 157473356 157473609 254 browser details YourSeq 153 2175 2378 3000 90.6% chr15 - 54059135 54059367 233 browser details YourSeq 152 2168 2379 3000 90.2% chr6 - 33860538 33860776 239 browser details YourSeq 152 2045 2378 3000 82.0% chr15 - 25311840 25312100 261 browser details YourSeq 150 2189 2375 3000 93.2% chr7 + 142877611 142877827 217 browser details YourSeq 149 2146 2367 3000 91.7% chr7 + 52011144 52011387 244 browser details YourSeq 148 2145 2377 3000 93.7% chr17 - 70684497 70684737 241 browser details YourSeq 147 2169 2378 3000 91.3% chr16 - 53040232 53040677 446 browser details YourSeq 147 2176 2377 3000 90.3% chr11 - 7852332 7852580 249 browser details YourSeq 146 2173 2378 3000 89.8% chr12 - 37029749 37029986 238 browser details YourSeq 145 2123 2378 3000 88.8% chr1 + 51670691 51670956 266 browser details YourSeq 144 2139 2346 3000 87.3% chr5 - 109813731 109813925 195 Note: The 3000 bp section upstream of Exon 7 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% chr14 + 25636269 25639268 3000 browser details YourSeq 82 976 1338 3000 94.8% chr11 - 114810791 114811226 436 browser details YourSeq 47 970 1032 3000 94.4% chr17 + 47207675 47208136 462 browser details YourSeq 44 976 1035 3000 95.9% chr6 - 125973587 125974127 541 browser details YourSeq 44 957 1015 3000 80.5% chr12 - 113128791 113128837 47 browser details YourSeq 41 970 1015 3000 95.7% chr9 - 64448756 64448802 47 browser details YourSeq 41 968 1019 3000 95.7% chr9 + 57414227 57414286 60 browser details YourSeq 41 970 1020 3000 97.7% chr2 + 90538058 90538109 52 browser details YourSeq 41 972 1021 3000 95.7% chr1 + 7029595 7029645 51 browser details YourSeq 39 966 1015 3000 90.0% chr1 - 36933346 36933396 51 browser details YourSeq 39 1016 1077 3000 95.5% chr19 + 45325901 45326063 163 browser details YourSeq 39 973 1021 3000 97.6% chr14 + 63108768 63108817 50 browser details YourSeq 39 970 1019 3000 97.6% chr11 + 76476877 76476928 52 browser details YourSeq 39 959 1021 3000 77.3% chr11 + 45860304 45860357 54 browser details YourSeq 38 971 1018 3000 97.5% chr9 - 60177999 60178047 49 browser details YourSeq 38 981 1021 3000 97.6% chr7 - 117949462 117949503 42 browser details YourSeq 38 981 1021 3000 97.6% chr7 - 76237005 76237046 42 browser details YourSeq 38 976 1016 3000 97.6% chr15 + 102286316 102286357 42 browser details YourSeq 37 970 1019 3000 93.1% chr9 - 109904853 109904903 51 browser details YourSeq 37 951 1014 3000 86.3% chr6 - 40668851 40668916 66 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Zmiz1 zinc finger, MIZ-type containing 1 [ Mus musculus (house mouse) ] Gene ID: 328365, updated on 12-Aug-2019 Gene summary Official Symbol Zmiz1 provided by MGI Official Full Name zinc finger, MIZ-type containing 1 provided by MGI Primary source MGI:MGI:3040693 See related Ensembl:ENSMUSG00000007817 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 Rai17; Zimp10; Gm10397; BC065120; E330020C23; I920194n01 Expression Ubiquitous expression in adrenal adult (RPKM 35.4), limb E14.5 (RPKM 30.1) and 27 other tissues See more Orthologs human all Genomic context Location: 14; 14 A3 See Zmiz1 in Genome Data Viewer Exon count: 27 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (25457875..25666743) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (26278778..26484205) Chromosome 14 - NC_000080.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Zmiz1 ENSMUSG00000007817 Description zinc finger, MIZ-type containing 1 [Source:MGI Symbol;Acc:MGI:3040693] Gene Synonyms Rai17, Zimp10 Location Chromosome 14: 25,459,185-25,666,743 forward strand. GRCm38:CM001007.2 About this gene This gene has 8 transcripts (splice variants), 197 orthologues, 5 paralogues, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Zmiz1-208 ENSMUST00000162645.7 7482 1072aa ENSMUSP00000124863.1 Protein coding CCDS26873 Q6P1E1 TSL:1 GENCODE basic APPRIS P3 Zmiz1-201 ENSMUST00000007961.14 5397 1066aa ENSMUSP00000007961.8 Protein coding CCDS79280 Q6P1E1 TSL:1 GENCODE basic APPRIS ALT1 Zmiz1-202 ENSMUST00000159766.7 4314 No protein - Retained intron - - TSL:2 Zmiz1-206 ENSMUST00000161766.7 2225 No protein - Retained intron - - TSL:1 Zmiz1-207 ENSMUST00000162546.1 460 No protein - Retained intron - - TSL:2 Zmiz1-204 ENSMUST00000160229.1 930 No protein - lncRNA - - TSL:3 Zmiz1-203 ENSMUST00000160190.1 359 No protein - lncRNA - - TSL:3 Zmiz1-205 ENSMUST00000160680.2 342 No protein - lncRNA - - TSL:5 Page 6 of 8 https://www.alphaknockout.com 227.56 kb Forward strand 25.45Mb 25.50Mb 25.55Mb 25.60Mb 25.65Mb Genes (Comprehensive set... Gm17747-201 >lncRNA Gm47921-201 >lncRNA Mir3075-201 >miRNA Zmiz1-203 >lncRNA Zmiz1-207 >retained intron Zmiz1-206 >retained intron Zmiz1-204 >lncRNA Zmiz1-208 >protein coding Zmiz1-201 >protein coding Zmiz1-202 >retained intron Gm47920-201 >lncRNA Zmiz1-205 >lncRNA Contigs AC157650.2 > < AC154455.2 Genes < Zmiz1os1-202lncRNA (Comprehensive set... < Zmiz1os1-201lncRNA Regulatory Build 25.45Mb 25.50Mb 25.55Mb 25.60Mb 25.65Mb Reverse strand 227.56 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: ENSMUST00000162645 207.52 kb Forward strand Zmiz1-208 >protein coding ENSMUSP00000124... MobiDB lite Low complexity (Seg) Pfam Zmiz1, N-terminal tetratricopeptide repeat domain Zinc finger, MIZ-type PROSITE profiles Zinc finger, MIZ-type PANTHER PTHR10782:SF7 PTHR10782 Gene3D Zinc finger, RING/FYVE/PHD-type CDD cd16791 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend frameshift variant splice region variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1072 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.