https://www.alphaknockout.com Mouse Zic3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Zic3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Zic3 gene (NCBI Reference Sequence: NM_009575 ; Ensembl: ENSMUSG00000067860 ) is located on Mouse chromosome X. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000088627). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Zic3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-420G11 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: Mutants show incomplete penetrance varying by genetic background and allele. Phenotypes range from bent tail/skeletal abnormalities to severe defects in embryo turning, cardiac development and neural tube closure resulting in death at embryonic day 18.5. Exon 1 covers 75.61% of the coding region. Start codon is in exon 1, and stop codon is in exon 3. The size of intron 1 for 3'-loxP site insertion: 1124 bp. The size of effective cKO region: ~1317 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele A T 5' G gRNA region 3' 1 2 3 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Zic3 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(7557bp) | A(23.18% 1752) | C(25.35% 1916) | T(27.25% 2059) | G(24.22% 1830) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chrX + 58028182 58031181 3000 browser details YourSeq 49 2748 2817 3000 94.8% chr10 + 122675202 122675281 80 browser details YourSeq 42 2749 2828 3000 67.4% chr13 - 52118926 52118971 46 browser details YourSeq 38 2749 2827 3000 69.1% chr10 - 122196528 122196575 48 browser details YourSeq 35 2749 2820 3000 67.5% chr13 - 44454690 44454737 48 browser details YourSeq 33 2756 2819 3000 97.3% chr15 + 51641636 51641703 68 browser details YourSeq 32 2747 2819 3000 77.2% chr18 - 59962613 59962674 62 browser details YourSeq 31 2749 2819 3000 64.8% chr16 - 87579227 87579265 39 browser details YourSeq 31 2749 2819 3000 60.7% chr18 + 72687215 72687247 33 browser details YourSeq 29 2757 2793 3000 87.9% chr11 + 104975471 104975506 36 browser details YourSeq 26 2752 2778 3000 100.0% chr1_GL456213_random + 19225 19253 29 browser details YourSeq 26 2749 2778 3000 89.3% chr1 + 135224991 135225019 29 browser details YourSeq 25 2745 2778 3000 75.0% chr14 + 76062419 76062447 29 browser details YourSeq 24 2749 2774 3000 96.2% chr1 - 81253579 81253604 26 browser details YourSeq 23 2755 2779 3000 87.5% chr15 + 53254266 53254289 24 browser details YourSeq 20 1126 1145 3000 100.0% chr17 + 10763346 10763365 20 Note: The 3000 bp section upstream of Exon 1 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% chrX + 58032489 58035488 3000 browser details YourSeq 118 871 1040 3000 84.8% chr14 + 122478537 122478706 170 browser details YourSeq 102 2491 2731 3000 77.2% chr3 - 99955578 99955808 231 browser details YourSeq 89 921 1041 3000 86.8% chr9 - 91362455 91362575 121 browser details YourSeq 79 841 979 3000 77.5% chr9 + 91384003 91384119 117 browser details YourSeq 24 1772 1807 3000 83.4% chr7 + 66984416 66984451 36 browser details YourSeq 21 1205 1245 3000 75.7% chr4 - 140855951 140855991 41 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Zic3 zinc finger protein of the cerebellum 3 [ Mus musculus (house mouse) ] Gene ID: 22773, updated on 12-Aug-2019 Gene summary Official Symbol Zic3 provided by MGI Official Full Name zinc finger protein of the cerebellum 3 provided by MGI Primary source MGI:MGI:106676 See related Ensembl:ENSMUSG00000067860 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 Bn; Ka Expression Biased expression in CNS E11.5 (RPKM 5.2), whole brain E14.5 (RPKM 4.7) and 7 other tissues See more Orthologs human all Genomic context Location: X A6; X 32.56 cM See Zic3 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (58030628..58036630) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (55283805..55289807) Chromosome X - NC_000086.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Zic3 ENSMUSG00000067860 Description zinc finger protein of the cerebellum 3 [Source:MGI Symbol;Acc:MGI:106676] Gene Synonyms Ka Location Chromosome X: 58,022,700-58,041,736 forward strand. GRCm38:CM001013.2 About this gene This gene has 4 transcripts (splice variants), 183 orthologues, 13 paralogues, is a member of 1 Ensembl protein family and is associated with 107 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Zic3-201 ENSMUST00000088627.10 3949 466aa ENSMUSP00000085999.4 Protein coding CCDS30155 Q62521 TSL:1 GENCODE basic APPRIS P2 Zic3-203 ENSMUST00000088631.10 2980 246aa ENSMUSP00000086003.4 Protein coding - A2AWK6 TSL:5 GENCODE basic Zic3-202 ENSMUST00000088629.3 1951 456aa ENSMUSP00000086001.3 Protein coding - Q62521 TSL:1 GENCODE basic APPRIS ALT1 Zic3-204 ENSMUST00000137687.1 566 89aa ENSMUSP00000116383.1 Protein coding - A2AWK4 CDS 3' incomplete TSL:3 39.04 kb Forward strand 58.02Mb 58.03Mb 58.04Mb 58.05Mb Genes (Comprehensive set... Zic3-204 >protein coding Zic3-201 >protein coding Zic3-203 >protein coding Zic3-202 >protein coding Contigs AL672124.13 > AL954389.5 > AL671920.9 > Regulatory Build 58.02Mb 58.03Mb 58.04Mb 58.05Mb Reverse strand 39.04 kb Regulation Legend Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000088627 5.92 kb Forward strand Zic3-201 >protein coding ENSMUSP00000085... MobiDB lite Low complexity (Seg) Superfamily Zinc finger C2H2 superfamily SMART Zinc finger C2H2-type Pfam ZIC protein, zinc finger domain Zinc finger C2H2-type PROSITE profiles Zinc finger C2H2-type PROSITE patterns Zinc finger C2H2-type PANTHER PTHR19818:SF25 PTHR19818 Gene3D 3.30.160.60 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 466 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.