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Mouse Ankrd27 Knockout Project (CRISPR/Cas9)

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Mouse Ankrd27 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ankrd27 Knockout Project (CRISPR/Cas9) Objective: To create a Ankrd27 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ankrd27 gene (NCBI Reference Sequence: NM_145633 ; Ensembl: ENSMUSG00000034867 ) is located on Mouse chromosome 7. 29 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 29 (Transcript: ENSMUST00000040844). Exon 3~11 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 3.28% of the coding region. Exon 3~11 covers 28.02% of the coding region. The size of effective KO region: ~6865 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 6 7 8 9 10 11 29 Legends Exon of mouse Ankrd27 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. 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 1141 bp section downstream of Exon 11 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(28.3% 566) | C(20.6% 412) | T(27.2% 544) | G(23.9% 478) 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(1141bp) | A(24.54% 280) | C(22.17% 253) | T(26.64% 304) | G(26.64% 304) Note: The 1141 bp section downstream of Exon 11 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% chr7 + 35598316 35600315 2000 browser details YourSeq 101 546 996 2000 79.6% chr10 + 41946957 41947229 273 browser details YourSeq 90 1095 1694 2000 83.1% chr3 - 94755439 94991486 236048 browser details YourSeq 75 1590 1740 2000 78.5% chr13 + 65515760 65515906 147 browser details YourSeq 72 899 1031 2000 87.4% chr7 - 123905691 123905821 131 browser details YourSeq 71 545 992 2000 79.5% chr10 + 3425167 3425556 390 browser details YourSeq 67 1638 1732 2000 87.7% chr11 - 89191854 89191946 93 browser details YourSeq 67 596 998 2000 94.9% chr11 + 84469330 84469893 564 browser details YourSeq 64 351 618 2000 78.6% chr12 + 85079576 85079801 226 browser details YourSeq 59 1615 1696 2000 92.9% chr15 + 89433787 89433879 93 browser details YourSeq 58 901 1126 2000 79.1% chr5 + 147141451 147141635 185 browser details YourSeq 55 900 997 2000 91.9% chr4 - 41836293 41836389 97 browser details YourSeq 55 900 1067 2000 76.2% chr11 - 53438407 53438537 131 browser details YourSeq 54 900 996 2000 91.7% chr4 - 41994557 41994652 96 browser details YourSeq 54 900 996 2000 91.7% chr4 - 42346772 42346867 96 browser details YourSeq 53 1643 1697 2000 98.2% chrX - 102490588 102490642 55 browser details YourSeq 53 1641 1695 2000 98.2% chr18 - 65275931 65275985 55 browser details YourSeq 53 1643 1737 2000 85.6% chr18 - 36484474 36484567 94 browser details YourSeq 52 899 994 2000 91.4% chr2 + 30922410 30922504 95 browser details YourSeq 52 902 997 2000 87.1% chr13 + 63686029 63686122 94 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 1141 1 1141 1141 100.0% chr7 + 35607181 35608321 1141 browser details YourSeq 92 510 741 1141 87.9% chr13 + 85481770 85482007 238 browser details YourSeq 87 661 776 1141 92.4% chr14 + 81001227 81001467 241 browser details YourSeq 85 530 848 1141 80.2% chr13 - 99604990 99605286 297 browser details YourSeq 81 496 850 1141 78.8% chr11 - 85045291 85045587 297 browser details YourSeq 81 576 736 1141 92.8% chr1 + 132408973 132409246 274 browser details YourSeq 73 562 690 1141 90.2% chr18 - 7738565 7738699 135 browser details YourSeq 72 490 711 1141 88.9% chr13 - 114984497 114984717 221 browser details YourSeq 70 530 854 1141 91.8% chr13 - 78088928 78089306 379 browser details YourSeq 69 575 829 1141 75.0% chr14 - 55839713 55839902 190 browser details YourSeq 67 573 831 1141 73.7% chr4 - 22058884 22059089 206 browser details YourSeq 65 557 833 1141 72.3% chr8 + 116269339 116269523 185 browser details YourSeq 64 717 850 1141 83.9% chr19 + 53707596 53707709 114 browser details YourSeq 63 560 882 1141 68.7% chr15 + 97524071 97524208 138 browser details YourSeq 62 573 850 1141 78.6% chr14 + 75486947 75487181 235 browser details YourSeq 61 557 651 1141 88.8% chr15 - 89154781 89154877 97 browser details YourSeq 61 510 845 1141 69.8% chr12 + 74135901 74136167 267 browser details YourSeq 60 510 651 1141 94.2% chr10 - 18678214 18678699 486 browser details YourSeq 60 554 755 1141 77.5% chr8 + 128315314 128315471 158 browser details YourSeq 59 530 1029 1141 66.2% chr3 + 51482317 51482404 88 Note: The 1141 bp section downstream of Exon 11 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Ankrd27 ankyrin repeat domain 27 (VPS9 domain) [ Mus musculus (house mouse) ] Gene ID: 245886, updated on 12-Aug-2019 Gene summary Official Symbol Ankrd27 provided by MGI Official Full Name ankyrin repeat domain 27 (VPS9 domain) provided by MGI Primary source MGI:MGI:2444103 See related Ensembl:ENSMUSG00000034867 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 Varp; AA408090; BC016493; D330003H11Rik Expression Ubiquitous expression in limb E14.5 (RPKM 10.8), bladder adult (RPKM 7.7) and 28 other tissues See more Orthologs human all Genomic context Location: 7; 7 B2 See Ankrd27 in Genome Data Viewer Exon count: 29 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (35586226..35639237) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (36371266..36424256) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Ankrd27 ENSMUSG00000034867 Description ankyrin repeat domain 27 (VPS9 domain) [Source:MGI Symbol;Acc:MGI:2444103] Gene Synonyms D330003H11Rik, Varp Location Chromosome 7: 35,586,244-35,639,226 forward strand. GRCm38:CM001000.2 About this gene This gene has 10 transcripts (splice variants), 213 orthologues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ankrd27- ENSMUST00000040844.15 4284 1048aa ENSMUSP00000041751.9 Protein coding CCDS21154 Q3UMR0 TSL:1 201 GENCODE basic APPRIS P2 Ankrd27- ENSMUST00000186245.6 1926 377aa ENSMUSP00000140554.1 Protein coding CCDS80706 A0A0R4J2C4 TSL:1 202 GENCODE basic Ankrd27- ENSMUST00000190503.6 4086 993aa ENSMUSP00000140259.1 Protein coding - Q3UMR0 TSL:1 206 GENCODE basic APPRIS ALT2 Ankrd27- ENSMUST00000187807.1 693 231aa ENSMUSP00000140848.1 Protein coding - A0A087WS06 CDS 5' and 3' 204 incomplete TSL:5 Ankrd27- ENSMUST00000188906.6 405 85aa ENSMUSP00000139753.2 Protein coding - A0A087WPF2 CDS 3' 205 incomplete TSL:3 Ankrd27- ENSMUST00000206472.1 3583 388aa ENSMUSP00000146118.1 Nonsense mediated - A0A0U1RPT7 TSL:5 209 decay Ankrd27- ENSMUST00000206157.1 797 76aa ENSMUSP00000145969.1 Nonsense mediated - A0A0U1RPG1 TSL:5 208 decay Ankrd27- ENSMUST00000206632.1 475 23aa ENSMUSP00000145817.1 Nonsense mediated - A0A0U1RP40 CDS 5' 210 decay incomplete TSL:3 Ankrd27- ENSMUST00000205801.1 699 No - Retained intron - - TSL:2 207 protein Ankrd27- ENSMUST00000187567.1 551 No - Retained intron - - TSL:3 203 protein Page 7 of 9 https://www.alphaknockout.com 72.98 kb Forward strand 35.58Mb 35.60Mb 35.62Mb 35.64Mb Genes (Comprehensive set... Ankrd27-201 >protein coding Ankrd27-202 >protein coding Ankrd27-203 >retained intron Ankrd27-205 >protein coding Ankrd27-204 >protein coding Ankrd27-209 >nonsense mediated decay Ankrd27-208 >nonsense mediated decay Ankrd27-210 >nonsense mediated decay Ankrd27-206 >protein coding Ankrd27-207 >retained intron Contigs < AC151531.3 Genes < Rgs9bp-201protein coding < Pdcd5-207retained intron (Comprehensive set... < Pdcd5-203retained intron < Pdcd5-204retained intron < Pdcd5-205retained intron < Pdcd5-206retained intron < Pdcd5-201protein coding < Pdcd5-202protein coding Regulatory Build 35.58Mb 35.60Mb 35.62Mb 35.64Mb Reverse strand 72.98 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000040844 52.98 kb Forward strand Ankrd27-201 >protein coding ENSMUSP00000041..
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