https://www.alphaknockout.com Mouse Zcchc2 Knockout Project (CRISPR/Cas9) Objective: To create a Zcchc2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Zcchc2 gene (NCBI Reference Sequence: NM_001122675 ; Ensembl: ENSMUSG00000038866 ) is located on Mouse chromosome 1. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000119166). Exon 3~5 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: Exon 3 starts from about 29.75% of the coding region. Exon 3~5 covers 7.56% of the coding region. The size of effective KO region: ~9824 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 13 Legends Exon of mouse Zcchc2 Knockout region Page 2 of 9 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 Exon 3 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 Exon 5 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.4% 488) | C(23.25% 465) | T(32.95% 659) | G(19.4% 388) Note: The 2000 bp section upstream of Exon 3 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.9% 538) | C(18.8% 376) | T(24.45% 489) | G(29.85% 597) Note: The 2000 bp section downstream of Exon 5 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 9 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% chr1 + 106002101 106004100 2000 browser details YourSeq 64 1498 1571 2000 97.2% chr11 - 6215317 6215402 86 browser details YourSeq 59 1244 1568 2000 69.5% chr6 + 82424034 82424137 104 browser details YourSeq 58 1445 1568 2000 94.1% chr5 - 26594081 26594233 153 browser details YourSeq 57 1504 1566 2000 96.9% chr13 - 114049381 114049448 68 browser details YourSeq 57 1505 1591 2000 93.9% chr14 + 75749600 75750105 506 browser details YourSeq 57 1517 1587 2000 88.9% chr14 + 34492236 34492303 68 browser details YourSeq 56 1505 1584 2000 95.2% chr7 + 144307145 144307233 89 browser details YourSeq 56 1503 1582 2000 90.8% chr18 + 62795340 62795418 79 browser details YourSeq 56 1504 1588 2000 88.9% chr16 + 44330855 44330936 82 browser details YourSeq 55 1510 1587 2000 92.2% chr4 - 142700959 142701036 78 browser details YourSeq 53 1504 1577 2000 94.9% chr11 + 72821442 72821518 77 browser details YourSeq 52 1504 1588 2000 77.6% chrX + 38887631 38887690 60 browser details YourSeq 52 1504 1568 2000 90.0% chr2 + 163343871 163343934 64 browser details YourSeq 51 1504 1562 2000 95.0% chr12 + 112640277 112640340 64 browser details YourSeq 50 1502 1566 2000 93.3% chr9 + 107990896 107990965 70 browser details YourSeq 50 1508 1568 2000 91.9% chr1 + 34390722 34390786 65 browser details YourSeq 49 1504 1565 2000 96.3% chrX + 164231754 164231817 64 browser details YourSeq 49 1505 1560 2000 98.1% chr17 + 32937850 32937910 61 browser details YourSeq 48 1501 1591 2000 98.1% chr11 + 3673169 3673261 93 Note: The 2000 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 2000 1 2000 2000 100.0% chr1 + 106011816 106013815 2000 browser details YourSeq 50 687 775 2000 94.6% chr1 + 33726408 33726838 431 browser details YourSeq 44 709 775 2000 91.7% chr6 - 38521405 38521470 66 browser details YourSeq 44 692 771 2000 84.4% chr7 + 3165586 3165673 88 browser details YourSeq 43 714 775 2000 95.8% chr3 - 112628625 112628694 70 browser details YourSeq 42 691 761 2000 92.0% chr1 + 167260820 167260932 113 browser details YourSeq 40 714 771 2000 97.7% chr5 + 48603556 48603621 66 browser details YourSeq 39 704 760 2000 95.3% chr3 + 90473207 90473270 64 browser details YourSeq 39 692 760 2000 86.8% chr11 + 14095726 14095802 77 browser details YourSeq 36 714 775 2000 95.0% chr18 + 67777222 67777287 66 browser details YourSeq 34 696 775 2000 87.0% chr8 - 69904906 69904986 81 browser details YourSeq 33 709 802 2000 97.2% chr1 + 64957396 64957669 274 browser details YourSeq 32 689 730 2000 88.1% chr15 - 80828875 80828916 42 browser details YourSeq 32 689 774 2000 67.6% chr11 + 94306979 94307062 84 browser details YourSeq 31 692 728 2000 91.9% chr7 - 122094360 122094396 37 browser details YourSeq 31 709 761 2000 91.9% chr16 - 10158411 10158471 61 browser details YourSeq 31 746 778 2000 97.0% chr11 + 95650086 95650118 33 browser details YourSeq 30 748 777 2000 100.0% chr2 + 73800611 73800640 30 browser details YourSeq 29 692 728 2000 96.8% chr3 - 88605269 88605306 38 browser details YourSeq 29 692 728 2000 89.2% chr15 - 81808805 81808841 37 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Zcchc2 zinc finger, CCHC domain containing 2 [ Mus musculus (house mouse) ] Gene ID: 227449, updated on 12-Aug-2019 Gene summary Official Symbol Zcchc2 provided by MGI Official Full Name zinc finger, CCHC domain containing 2 provided by MGI Primary source MGI:MGI:2444114 See related Ensembl:ENSMUSG00000038866 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 AW212015; 9930114B20Rik Expression Ubiquitous expression in testis adult (RPKM 19.4), thymus adult (RPKM 8.8) and 28 other tissues See more Orthologs human all Genomic context Location: 1; 1 E2.1 See Zcchc2 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (105990390..106034077) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (107886983..107930654) Chromosome 1 - NC_000067.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Zcchc2 ENSMUSG00000038866 Description zinc finger, CCHC domain containing 2 [Source:MGI Symbol;Acc:MGI:2444114] Gene Synonyms 9930114B20Rik Location Chromosome 1: 105,990,406-106,034,074 forward strand. GRCm38:CM000994.2 About this gene This gene has 5 transcripts (splice variants), 212 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Zcchc2- ENSMUST00000119166.7 6368 1155aa ENSMUSP00000113128.1 Protein coding CCDS48336 Q69ZB8 TSL:1 202 GENCODE basic APPRIS ALT2 Zcchc2- ENSMUST00000118196.7 5796 1166aa ENSMUSP00000113974.1 Protein coding CCDS48335 Q69ZB8 TSL:5 201 GENCODE basic APPRIS P4 Zcchc2- ENSMUST00000186983.6 3455 758aa ENSMUSP00000141148.1 Protein coding - A0A087WSQ9 CDS 5' 204 incomplete TSL:1 Zcchc2- ENSMUST00000188954.6 770 257aa ENSMUSP00000139971.1 Protein coding - A0A087WPY9 CDS 5' and 3' 205 incomplete TSL:3 Zcchc2- ENSMUST00000131830.2 601 79aa ENSMUSP00000140540.1 Nonsense mediated - A0A087WRA5 CDS 5' 203 decay incomplete TSL:3 Page 7 of 9 https://www.alphaknockout.com 63.67 kb Forward strand 105.99Mb 106.00Mb 106.01Mb 106.02Mb 106.03Mb 106.04Mb Genes (Comprehensive set... Zcchc2-201 >protein coding Zcchc2-202 >protein coding Zcchc2-204 >protein coding Zcchc2-205 >protein coding Zcchc2-203 >nonsense mediated decay Contigs < AC132123.3 AC099640.6 > Genes < Gm7160-201lncRNA (Comprehensive set... < Gm7160-202lncRNA Regulatory Build 105.99Mb 106.00Mb 106.01Mb 106.02Mb 106.03Mb 106.04Mb Reverse strand 63.67 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000119166 43.38 kb Forward strand Zcchc2-202 >protein coding ENSMUSP00000113... MobiDB lite Low complexity (Seg) Superfamily PX domain superfamily Zinc finger, CCHC-type superfamily SMART Zinc finger, CCHC-type Pfam Zinc finger, CCHC-type PROSITE profiles Zinc finger, CCHC-type PANTHER PTHR46939 Gene3D PX domain superfamily 4.10.60.10 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion missense variant splice region variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1155 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.