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Mouse Ckap4 Knockout Project (CRISPR/Cas9)

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Mouse Ckap4 Knockout Project (CRISPR/Cas9) http://www.alphaknockout.com/ Mouse Ckap4 Knockout Project (CRISPR/Cas9) Objective: To create a Ckap4 knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ckap4 gene ( NCBI Reference Sequence: NM_175451 ; Ensembl: ENSMUSG00000046841 ) is located on mouse chromosome 10. 2 exons are identified , with the ATG start codon in exon 1 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000053871). Exon 2 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 2 starts from about 24.41% of the coding region. Exon 2 covers 75.65% of the coding region. The size of effective KO region: ~3243 bp. Page 1 of 8 http://www.alphaknockout.com/ Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 Legends Exon of mouse Ckap4 Knockout region Page 2 of 8 http://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 2 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 2 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 http://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(22.15% 443) | G(25.1% 502) | T(28.35% 567) Note: The 2000 bp section upstream of Exon 2 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(25.6% 512) | C(21.65% 433) | G(24.45% 489) | T(28.3% 566) Note: The 2000 bp section downstream of Exon 2 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 http://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% chr10 - 84528778 84530777 2000 browser details YourSeq 75 448 784 2000 71.2% chr2 + 87442813 87443040 228 browser details YourSeq 74 702 797 2000 91.3% chr11 - 95404419 95404517 99 browser details YourSeq 74 607 782 2000 80.6% chr1 + 16556657 16556830 174 browser details YourSeq 73 600 783 2000 82.5% chr12 - 75295217 75295399 183 browser details YourSeq 68 606 783 2000 79.2% chr1 - 66215439 66215626 188 browser details YourSeq 66 414 775 2000 82.9% chr11 + 103990875 103991403 529 browser details YourSeq 65 702 783 2000 90.3% chr18 + 38457090 38457172 83 browser details YourSeq 65 706 785 2000 91.3% chr1 + 159086240 159086320 81 browser details YourSeq 63 702 782 2000 90.2% chr14 - 27011196 27011732 537 browser details YourSeq 63 700 792 2000 91.2% chr10 - 105678101 105678199 99 browser details YourSeq 63 693 782 2000 82.8% chr7 + 144653073 144653160 88 browser details YourSeq 62 702 782 2000 88.9% chr4 - 103693300 103693381 82 browser details YourSeq 62 702 787 2000 86.1% chr12 + 84776748 84776833 86 browser details YourSeq 61 702 782 2000 87.7% chr1 - 34520881 34520961 81 browser details YourSeq 61 702 788 2000 85.1% chr16 + 8832882 8832968 87 browser details YourSeq 60 702 782 2000 87.7% chr14 - 65249154 65249235 82 browser details YourSeq 60 686 783 2000 84.9% chr10 + 92789793 92789905 113 browser details YourSeq 59 608 763 2000 85.6% chr11 - 114195919 114196075 157 browser details YourSeq 59 702 778 2000 88.4% chr12 + 69750461 69750537 77 Note: The 2000 bp section upstream of Exon 2 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% chr10 - 84524305 84526304 2000 browser details YourSeq 258 735 1154 2000 84.6% chr18 + 11551328 11551752 425 browser details YourSeq 242 734 1172 2000 85.9% chr11 + 94061921 94062357 437 browser details YourSeq 237 783 1153 2000 87.2% chr18 - 78083532 78083910 379 browser details YourSeq 223 735 1154 2000 85.1% chr15 - 79321780 79371390 49611 browser details YourSeq 220 735 1136 2000 83.0% chr17 - 79294268 79294678 411 browser details YourSeq 218 735 1117 2000 86.9% chr9 - 37274082 37274464 383 browser details YourSeq 218 783 1174 2000 88.1% chr1 + 19748532 19748935 404 browser details YourSeq 217 737 1135 2000 83.7% chr5 - 120775753 120776153 401 browser details YourSeq 215 788 1154 2000 85.4% chr1 + 132719900 132720263 364 browser details YourSeq 214 786 1135 2000 89.7% chr13 - 100888790 100889153 364 browser details YourSeq 213 799 1202 2000 84.4% chr18 - 49657577 49658003 427 browser details YourSeq 209 735 1122 2000 86.3% chr8 - 45347731 45348125 395 browser details YourSeq 209 734 1206 2000 78.2% chr16 + 12895743 12896250 508 browser details YourSeq 208 781 1136 2000 85.9% chr17 + 56152573 56152934 362 browser details YourSeq 207 781 1145 2000 82.9% chr15 + 43417867 43418228 362 browser details YourSeq 204 785 1200 2000 85.4% chr7 + 23449014 23449431 418 browser details YourSeq 203 778 1116 2000 87.9% chr18 + 65199550 65199895 346 browser details YourSeq 199 781 1154 2000 85.0% chr13 + 101603674 101604047 374 browser details YourSeq 197 781 1159 2000 86.4% chr6 + 18554966 18555363 398 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 http://www.alphaknockout.com/ Gene and protein information: Ckap4 cytoskeleton-associated protein 4 [ Mus musculus (house mouse) ] Gene ID: 216197, updated on 17-Nov-2020 Gene summary Official Symbol Ckap4 provided by MGI Official Full Name cytoskeleton-associated protein 4 provided by MGI Primary source MGI:MGI:2444926 See related Ensembl:ENSMUSG00000046841 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 P6; P63; CLIMP; CLIMP-63; 5630400A09Rik Expression Broad expression in ovary adult (RPKM 52.9), limb E14.5 (RPKM 52.2) and 27 other tissues See more Orthologs human all Genomic context Location: 10; 10 C1 See Ckap4 in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 109 current GRCm39 (GCF_000001635.27) 10 NC_000076.7 (84362169..84369752, complement) 108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (84526305..84533888, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (83989050..83996633, complement) Chromosome 10 - NC_000076.7 Page 6 of 8 http://www.alphaknockout.com/ Transcript information: This gene has 2 transcripts Gene: Ckap4 ENSMUSG00000046841 Description cytoskeleton-associated protein 4 [Source:MGI Symbol;Acc:MGI:2444926] Gene Synonyms 5630400A09Rik, CLIMP-63, P63 Location Chromosome 10: 84,526,305-84,534,062 reverse strand. GRCm38:CM001003.2 About this gene This gene has 2 transcripts (splice variants), 262 orthologues, 7 paralogues and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Match Flags Ckap4-201 ENSMUST00000053871.4 3089 575aa ENSMUSP00000050336.3 Protein coding CCDS24080 Q8BMK4 TSL:1 GENCODE basic APPRIS P1 Ckap4-202 ENSMUST00000167671.1 2606 575aa ENSMUSP00000130304.1 Protein coding CCDS24080 Q8BMK4 TSL:1 GENCODE basic APPRIS P1 27.76 kb Forward strand 84.52Mb 84.53Mb 84.54Mb Genes E230014E18Rik-201 >lincRNA (Comprehensive set... Contigs AC150314.5 > < AC140333.2 Genes (Comprehensive set... < Ckap4-201protein coding < Ckap4-202protein coding Regulatory Build 84.52Mb 84.53Mb 84.54Mb Reverse strand 27.76 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene Page 7 of 8 http://www.alphaknockout.com/ Transcript: ENSMUST00000053871 < Ckap4-201protein coding Reverse strand 7.76 kb ENSMUSP00000050... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57997 PANTHER Cytoskeleton-associated protein 4 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 575 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC, VectorBuilder. Page 8 of 8.
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