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Mouse Akt1s1 Knockout Project (CRISPR/Cas9)

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Mouse Akt1s1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Akt1s1 Knockout Project (CRISPR/Cas9) Objective: To create a Akt1s1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Akt1s1 gene (NCBI Reference Sequence: NM_001290694 ; Ensembl: ENSMUSG00000011096 ) is located on Mouse chromosome 7. 5 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 5 (Transcript: ENSMUST00000107882). 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: Mice homozygous for a knock-out allele exhibit increased susceptibility to induced ischemic brain injury. Exon 2 starts from about 8.48% of the coding region. Exon 2~5 covers 91.64% of the coding region. The size of effective KO region: ~2162 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 2 3 4 5 Legends Exon of mouse Akt1s1 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 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 stop codon 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(23.25% 465) | C(23.45% 469) | T(24.25% 485) | G(29.05% 581) 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(23.45% 469) | C(24.95% 499) | T(25.7% 514) | G(25.9% 518) 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 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 + 44850572 44852571 2000 browser details YourSeq 58 1321 1677 2000 74.6% chr17 + 35560405 35605226 44822 browser details YourSeq 37 1240 1338 2000 91.2% chr11 - 99437030 99437129 100 browser details YourSeq 33 1321 1381 2000 97.2% chr5 - 106971520 106971718 199 browser details YourSeq 28 440 474 2000 96.7% chr18 + 19510636 19510683 48 browser details YourSeq 26 1321 1346 2000 100.0% chr7 - 28445138 28445163 26 browser details YourSeq 25 1715 1742 2000 84.7% chrX - 123582651 123582676 26 browser details YourSeq 25 1715 1742 2000 84.7% chrX - 123794616 123794641 26 browser details YourSeq 25 1715 1742 2000 84.7% chrX - 124022386 124022411 26 browser details YourSeq 25 1302 1342 2000 80.5% chr7 - 98693931 98693971 41 browser details YourSeq 25 1320 1344 2000 100.0% chr17 - 83463795 83463819 25 browser details YourSeq 25 1321 1345 2000 100.0% chr17 - 24029424 24029448 25 browser details YourSeq 25 1321 1346 2000 100.0% chr1 + 138563126 138563152 27 browser details YourSeq 25 1320 1344 2000 100.0% chr1 + 59308522 59308546 25 browser details YourSeq 25 1229 1256 2000 96.5% chr1 + 3856379 3856408 30 browser details YourSeq 24 1255 1278 2000 100.0% chr3 - 66371119 66371142 24 browser details YourSeq 23 1321 1343 2000 100.0% chr8 - 3444214 3444236 23 browser details YourSeq 23 1322 1344 2000 100.0% chr7 - 4418991 4419013 23 browser details YourSeq 23 1320 1342 2000 100.0% chr19 - 53014816 53014838 23 browser details YourSeq 23 1320 1342 2000 100.0% chr18 - 8877276 8877298 23 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% chr7 + 44854734 44856733 2000 browser details YourSeq 74 1408 1662 2000 96.3% chr5 - 145263902 145264442 541 browser details YourSeq 73 929 1507 2000 76.5% chr11 - 58227897 58228400 504 browser details YourSeq 72 1291 1507 2000 91.9% chr11 + 110213639 110214008 370 browser details YourSeq 67 1269 1453 2000 90.4% chr10 + 121332361 121332628 268 browser details YourSeq 67 1407 1507 2000 91.4% chr1 + 74722951 74723556 606 browser details YourSeq 66 1411 1507 2000 90.3% chr5 - 134019398 134019497 100 browser details YourSeq 66 1408 1507 2000 84.6% chr11 - 70426341 70426442 102 browser details YourSeq 66 1409 1505 2000 94.6% chr1 - 176252178 176252277 100 browser details YourSeq 65 1376 1507 2000 97.2% chr12 + 108464955 108465138 184 browser details YourSeq 65 1393 1507 2000 93.4% chr10 + 91184018 91184132 115 browser details YourSeq 64 1421 1507 2000 95.8% chr4 + 148084907 148084996 90 browser details YourSeq 63 1408 1507 2000 94.3% chr4 - 132643683 132643787 105 browser details YourSeq 63 1408 1503 2000 93.2% chr19 - 47835273 47835373 101 browser details YourSeq 63 1421 1507 2000 95.7% chr1 - 191957761 191957850 90 browser details YourSeq 63 1409 1951 2000 68.0% chr8 + 108570501 108570643 143 browser details YourSeq 62 1408 1507 2000 97.0% chr6 - 142787161 142787262 102 browser details YourSeq 62 1409 1503 2000 93.1% chr2 + 91648451 91648548 98 browser details YourSeq 62 1408 1507 2000 95.6% chr19 + 22555989 22556091 103 browser details YourSeq 61 1410 1507 2000 93.1% chr16 - 32103127 32103224 98 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Akt1s1 AKT1 substrate 1 (proline-rich) [ Mus musculus (house mouse) ] Gene ID: 67605, updated on 14-Aug-2019 Gene summary Official Symbol Akt1s1 provided by MGI Official Full Name AKT1 substrate 1 (proline-rich) provided by MGI Primary source MGI:MGI:1914855 See related Ensembl:ENSMUSG00000011096 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 Lobe; Lobel; PRAS40; AI227026; 1110012J22Rik Expression Ubiquitous expression in adrenal adult (RPKM 52.6), ovary adult (RPKM 41.8) and 28 other tissues See more Orthologs human all Genomic context Location: 7; 7 B3 See Akt1s1 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (44848961..44855421) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (52104597..52110780) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 12 transcripts Gene: Akt1s1 ENSMUSG00000011096 Description AKT1 substrate 1 (proline-rich) [Source:MGI Symbol;Acc:MGI:1914855] Gene Synonyms 1110012J22Rik, Lobe, PRAS40 Location Chromosome 7: 44,848,991-44,855,421 forward strand. GRCm38:CM001000.2 About this gene This gene has 12 transcripts (splice variants), 150 orthologues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Akt1s1- ENSMUST00000107880.8 1757 257aa ENSMUSP00000103512.1 Protein CCDS21218 Q9D1F4 TSL:1 202 coding GENCODE basic APPRIS P3 Akt1s1- ENSMUST00000054343.14 1592 257aa ENSMUSP00000049764.8 Protein CCDS21218 Q9D1F4 TSL:1 201 coding GENCODE basic APPRIS P3 Akt1s1- ENSMUST00000107882.7 1160 283aa ENSMUSP00000103514.1 Protein CCDS71954 E9QKI5 TSL:3 203 coding GENCODE basic APPRIS ALT2 Akt1s1- ENSMUST00000107885.7 1812 328aa ENSMUSP00000103517.1 Protein - E9QKI4 TSL:1 204 coding GENCODE basic APPRIS ALT2 Akt1s1- ENSMUST00000141311.7 835 193aa ENSMUSP00000120690.1 Protein - D3YWZ1 CDS 3' incomplete 207 coding TSL:2 Akt1s1- ENSMUST00000127783.1 750 247aa ENSMUSP00000116149.2 Protein - F6R8S6 CDS 3' incomplete 205 coding TSL:5 Akt1s1- ENSMUST00000150335.1 485 123aa ENSMUSP00000122607.1 Protein - D3Z4R0 CDS 3' incomplete 209 coding TSL:2 Akt1s1- ENSMUST00000208384.1 392 57aa ENSMUSP00000147235.1 Protein - A0A140LJJ3 CDS 3' incomplete 212 coding TSL:2 Akt1s1- ENSMUST00000142880.2 333 111aa ENSMUSP00000146332.1 Protein - A0A140LHA0 CDS 5' and 3' 208 coding incomplete TSL:3 Akt1s1- ENSMUST00000136232.1 332 95aa ENSMUSP00000116541.1 Protein - D3Z4W3 CDS 3' incomplete 206 coding TSL:2 Akt1s1- ENSMUST00000207223.1 212 14aa ENSMUSP00000146484.1 Protein - A0A140LHN7 CDS 3' incomplete 211 coding TSL:3 Akt1s1- ENSMUST00000152091.7 2207 No - Retained - - TSL:1 210 protein intron 26.43 kb Forward strand 44.84Mb 44.85Mb 44.86Mb Genes (Comprehensive set... Il4i1-202 >protein coding Akt1s1-204 >protein coding Pnkp-203 >protein coding
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