https://www.alphaknockout.com Mouse Zc3h12a Knockout Project (CRISPR/Cas9) Objective: To create a Zc3h12a knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Zc3h12a gene (NCBI Reference Sequence: NM_153159 ; Ensembl: ENSMUSG00000042677 ) is located on Mouse chromosome 4. 6 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000036188). Exon 2~6 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: Mice homozygous for a null allele exhibit splenomegaly, lymphadenopathy, hyperimmunoglobulinemia, increased auto-antibodies, and defective IL6 post-transcriptional regulation. Exon 2 starts from about 0.06% of the coding region. Exon 2~6 covers 100.0% of the coding region. The size of effective KO region: ~7765 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 2 3 4 5 6 Legends Exon of mouse Zc3h12a 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. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. 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(20.1% 402) | C(27.65% 553) | T(23.65% 473) | G(28.6% 572) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.4% 508) | C(26.2% 524) | T(22.2% 444) | G(26.2% 524) 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% chr4 - 125127049 125129048 2000 browser details YourSeq 138 160 321 2000 90.4% chr4 - 32919286 32919440 155 browser details YourSeq 134 158 306 2000 95.4% chr2 - 17012252 17012764 513 browser details YourSeq 133 160 305 2000 97.3% chr13 + 76507611 76507756 146 browser details YourSeq 133 161 306 2000 95.9% chr1 + 175377972 175378118 147 browser details YourSeq 133 160 306 2000 96.6% chr1 + 79731574 79731722 149 browser details YourSeq 132 160 305 2000 95.3% chr12 - 12407380 12407525 146 browser details YourSeq 132 160 306 2000 95.9% chr1 + 153494406 153494553 148 browser details YourSeq 131 165 307 2000 95.1% chr10 - 40215345 40215486 142 browser details YourSeq 131 160 305 2000 95.3% chr1 - 54982469 54982616 148 browser details YourSeq 131 171 516 2000 95.3% chr14 + 121407756 121408360 605 browser details YourSeq 130 163 298 2000 97.1% chr8 - 27125044 27125178 135 browser details YourSeq 130 160 302 2000 96.5% chr1 - 53988875 53989018 144 browser details YourSeq 130 159 302 2000 95.9% chr7 + 19189104 19189254 151 browser details YourSeq 130 165 306 2000 95.0% chr5 + 138158422 138158562 141 browser details YourSeq 130 160 305 2000 94.6% chr1 + 82780561 82780706 146 browser details YourSeq 129 160 298 2000 96.5% chr18 - 66390827 66390965 139 browser details YourSeq 129 161 302 2000 95.8% chr13 - 45305151 45305293 143 browser details YourSeq 129 160 305 2000 95.2% chr7 + 67991079 67991224 146 browser details YourSeq 129 160 305 2000 93.1% chr4 + 59327024 59327167 144 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% chr4 - 125117282 125119281 2000 browser details YourSeq 173 576 857 2000 91.8% chr7 - 110144717 110145260 544 browser details YourSeq 167 657 860 2000 93.3% chr12 - 22179781 22180051 271 browser details YourSeq 165 657 862 2000 89.9% chr5 - 122678452 122678643 192 browser details YourSeq 165 650 847 2000 90.2% chr5 + 30064552 30064745 194 browser details YourSeq 164 652 844 2000 94.6% chr8 - 43148812 43149005 194 browser details YourSeq 164 559 858 2000 91.8% chr11 - 51927440 51927790 351 browser details YourSeq 162 657 857 2000 89.5% chr4 + 41076050 41076243 194 browser details YourSeq 161 657 852 2000 91.7% chr3 - 31140435 31140638 204 browser details YourSeq 161 657 862 2000 89.8% chr12 - 21853004 21853201 198 browser details YourSeq 161 659 906 2000 93.1% chr1 + 159190491 159191098 608 browser details YourSeq 160 657 860 2000 91.2% chr11 + 78252861 78253065 205 browser details YourSeq 159 650 838 2000 93.1% chr16 + 8624580 8733096 108517 browser details YourSeq 159 657 842 2000 93.6% chr1 + 131878713 131878992 280 browser details YourSeq 158 655 844 2000 92.0% chr13 - 100211733 100211926 194 browser details YourSeq 158 657 859 2000 92.6% chr5 + 65516322 65516553 232 browser details YourSeq 158 657 859 2000 89.1% chr16 + 90256120 90256313 194 browser details YourSeq 157 657 856 2000 93.0% chr2 - 5945113 5945315 203 browser details YourSeq 157 652 856 2000 89.1% chr18 + 24439141 24439340 200 browser details YourSeq 157 657 834 2000 94.4% chr15 + 13330380 13330561 182 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: Zc3h12a zinc finger CCCH type containing 12A [ Mus musculus (house mouse) ] Gene ID: 230738, updated on 11-Sep-2019 Gene summary Official Symbol Zc3h12a provided by MGI Official Full Name zinc finger CCCH type containing 12A provided by MGI Primary source MGI:MGI:2385891 See related Ensembl:ENSMUSG00000042677 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 Reg1; MCPIP; Mcpip1; MCPIP-1; BC036563 Expression Broad expression in large intestine adult (RPKM 23.3), spleen adult (RPKM 14.6) and 15 other tissues See more Orthologs human all Genomic context Location: 4; 4 D2.2 See Zc3h12a in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (125118414..125127881, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (124795658..124805125, complement) Chromosome 4 - NC_000070.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Zc3h12a ENSMUSG00000042677 Description zinc finger CCCH type containing 12A [Source:MGI Symbol;Acc:MGI:2385891] Gene Synonyms Mcpip1 Location Chromosome 4: 125,118,423-125,127,840 reverse strand. GRCm38:CM000997.2 About this gene This gene has 2 transcripts (splice variants), 257 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 33 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Zc3h12a-201 ENSMUST00000036188.7 2755 596aa ENSMUSP00000037172.7 Protein coding CCDS18638 Q5D1E7 TSL:1 GENCODE basic APPRIS P1 Zc3h12a-202 ENSMUST00000131685.1 660 No protein - Retained intron - - TSL:1 29.42 kb Forward strand 125.11Mb 125.12Mb 125.13Mb Genes Meaf6-205 >protein coding (Comprehensive set... Meaf6-201 >protein coding Meaf6-208 >nonsense mediated decay Meaf6-202 >protein coding Meaf6-207 >nonsense mediated decay Meaf6-206 >retained intron Contigs AL626775.11 > Genes < Zc3h12a-201protein coding (Comprehensive set... < Zc3h12a-202retained intron Regulatory Build 125.11Mb 125.12Mb 125.13Mb Reverse strand 29.42 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000036188 < Zc3h12a-201protein coding Reverse strand 9.42 kb ENSMUSP00000037... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Pfam Rege-1, UBA-like domain Endoribonuclease Regnase 1/ZC3H12, C-terminal domain Ribonuclease Zc3h12a-like, NYN domain PANTHER PTHR12876:SF10 PTHR12876 Gene3D 3.40.50.11980 CDD cd18729 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 596 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.