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Mouse Akirin2 Knockout Project (CRISPR/Cas9)

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Mouse Akirin2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Akirin2 Knockout Project (CRISPR/Cas9) Objective: To create a Akirin2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Akirin2 gene (NCBI Reference Sequence: NM_001007589 ; Ensembl: ENSMUSG00000028291 ) is located on Mouse chromosome 4. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000084299). Exon 2~5 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: No homozygous null embryos are recovered at E9.5. Exon 2 starts from about 38.14% of the coding region. Exon 2~5 covers 62.02% of the coding region. The size of effective KO region: ~3911 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 Legends Exon of mouse Akirin2 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 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. 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. 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(31.65% 633) | C(14.55% 291) | T(34.6% 692) | G(19.2% 384) 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(29.9% 598) | C(18.5% 370) | T(32.6% 652) | G(19.0% 380) 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 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 + 34560313 34562312 2000 browser details YourSeq 78 2 124 2000 84.9% chr3 - 123887068 123887202 135 browser details YourSeq 75 1 115 2000 91.4% chr1 - 88440861 88441095 235 browser details YourSeq 70 2 113 2000 90.7% chr13 - 73975114 73975226 113 browser details YourSeq 69 2 110 2000 92.6% chr8 - 89213574 89213682 109 browser details YourSeq 68 1 106 2000 89.7% chrX - 23562551 23562772 222 browser details YourSeq 67 7 132 2000 75.5% chr11 - 32479585 32479702 118 browser details YourSeq 67 2 110 2000 91.4% chr18 + 56433691 56433799 109 browser details YourSeq 66 2 110 2000 91.3% chr8 - 40470319 40470428 110 browser details YourSeq 66 2 119 2000 91.2% chr9 + 87063315 87063438 124 browser details YourSeq 63 9 110 2000 92.0% chr3 - 28213966 28214068 103 browser details YourSeq 63 1 106 2000 93.3% chr6 + 86325940 86326227 288 browser details YourSeq 63 11 113 2000 93.2% chr12 + 18190660 18190764 105 browser details YourSeq 62 2 109 2000 93.3% chr7 - 83748315 83748423 109 browser details YourSeq 62 2 109 2000 93.1% chr12 - 117457002 117457111 110 browser details YourSeq 62 2 110 2000 88.9% chr8 + 104700661 104700771 111 browser details YourSeq 61 8 110 2000 89.1% chr7 - 107506241 107506342 102 browser details YourSeq 61 8 109 2000 91.9% chr2 - 153695045 153695148 104 browser details YourSeq 61 15 113 2000 93.0% chr14 - 123270698 123270797 100 browser details YourSeq 61 7 108 2000 91.9% chr1 - 164769423 164769526 104 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% chr4 + 34566224 34568223 2000 browser details YourSeq 55 1608 1747 2000 90.8% chr12 - 45826073 45826265 193 browser details YourSeq 55 1023 1101 2000 93.7% chr15 + 93992917 93993010 94 browser details YourSeq 50 1400 1756 2000 63.6% chr13 + 45399033 45399217 185 browser details YourSeq 49 1601 1762 2000 91.4% chr12 + 80671417 80671636 220 browser details YourSeq 45 1341 1748 2000 89.5% chr14 - 47601065 47601504 440 browser details YourSeq 39 1669 1728 2000 80.9% chr15 + 98707583 98707638 56 browser details YourSeq 37 1691 1755 2000 82.3% chr11 + 76016031 76016092 62 browser details YourSeq 35 1662 1747 2000 78.9% chr14 - 20774928 20775012 85 browser details YourSeq 35 1691 1749 2000 74.6% chr13 + 73941044 73941098 55 browser details YourSeq 33 1710 1756 2000 85.2% chr11 + 79322859 79322905 47 browser details YourSeq 32 1707 1752 2000 94.5% chr9 - 117358741 117358786 46 browser details YourSeq 32 1710 1762 2000 83.4% chr11 - 103912004 103912088 85 browser details YourSeq 32 1709 1752 2000 86.4% chr5 + 121716531 121716574 44 browser details YourSeq 32 1691 1747 2000 73.0% chr12 + 97854521 97854573 53 browser details YourSeq 31 1689 1743 2000 84.5% chr12 - 75678520 75678889 370 browser details YourSeq 31 1709 1747 2000 89.8% chr11 - 100068143 100068181 39 browser details YourSeq 31 1341 1377 2000 86.2% chr14 + 25810096 25810131 36 browser details YourSeq 31 1691 1747 2000 72.4% chr13 + 43334277 43334329 53 browser details YourSeq 31 1708 1748 2000 87.9% chr11 + 53256985 53257025 41 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Akirin2 akirin 2 [ Mus musculus (house mouse) ] Gene ID: 433693, updated on 12-Aug-2019 Gene summary Official Symbol Akirin2 provided by MGI Official Full Name akirin 2 provided by MGI Primary source MGI:MGI:1889364 See related Ensembl:ENSMUSG00000028291 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 AA114675; AA522011; AU019887; Akirin-2; 2700059D21Rik Expression Ubiquitous expression in CNS E14 (RPKM 44.4), whole brain E14.5 (RPKM 43.4) and 28 other tissues See more Orthologs human all Genomic context Location: 4; 4 A5 See Akirin2 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (34550615..34566965) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (34497864..34514157) Chromosome 4 - NC_000070.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Akirin2 ENSMUSG00000028291 Description akirin 2 [Source:MGI Symbol;Acc:MGI:1889364] Gene Synonyms 2700059D21Rik Location Chromosome 4: 34,550,937-34,566,908 forward strand. GRCm38:CM000997.2 About this gene This gene has 2 transcripts (splice variants), 248 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Akirin2-201 ENSMUST00000084299.5 1397 201aa ENSMUSP00000081322.5 Protein coding CCDS18027 B1AXD8 TSL:1 GENCODE basic APPRIS P1 Akirin2-202 ENSMUST00000132230.1 781 No protein - lncRNA - - TSL:3 35.97 kb Forward strand 34.55Mb 34.56Mb 34.57Mb Genes (Comprehensive set... Akirin2-201 >protein coding Akirin2-202 >lncRNA Contigs AL807397.6 > Genes < Orc3-202protein coding (Comprehensive set... < Orc3-201protein coding < Orc3-204lncRNA < Orc3-208lncRNA Regulatory Build 34.55Mb 34.56Mb 34.57Mb Reverse strand 35.97 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000084299 15.97 kb Forward strand Akirin2-201 >protein coding ENSMUSP00000081... Low complexity (Seg) Coiled-coils (Ncoils) PANTHER PTHR13293:SF8 Akirin All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 201 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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