https://www.alphaknockout.com

Mouse Akt1s1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Akt1s1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Akt1s1 (NCBI Reference Sequence: NM_001290694 ; Ensembl: ENSMUSG00000011096 ) is located on Mouse 7. 5 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 5 (Transcript: ENSMUST00000107882). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Akt1s1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-186N23 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO 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 3 starts from about 54.3% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 820 bp, and the size of intron 4 for 3'-loxP site insertion: 483 bp. The size of effective cKO region: ~820 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Akt1s1 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7311bp) | A(22.28% 1629) | C(25.95% 1897) | T(23.2% 1696) | G(28.57% 2089)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr7 + 44850531 44853530 3000 browser details YourSeq 58 1362 1718 3000 74.6% chr17 + 35560405 35605226 44822 browser details YourSeq 37 1281 1379 3000 91.2% chr11 - 99437030 99437129 100 browser details YourSeq 28 481 515 3000 96.7% chr18 + 19510636 19510683 48 browser details YourSeq 25 1362 1387 3000 100.0% chr1 + 138563126 138563152 27 browser details YourSeq 25 1361 1385 3000 100.0% chr1 + 59308522 59308546 25 browser details YourSeq 22 1362 1383 3000 100.0% chr19 + 47765660 47765681 22 browser details YourSeq 22 1362 1383 3000 100.0% chr17 + 86614065 86614086 22 browser details YourSeq 22 1361 1382 3000 100.0% chr17 + 29976560 29976581 22 browser details YourSeq 22 1363 1384 3000 100.0% chr1 + 82667415 82667436 22 browser details YourSeq 21 1362 1382 3000 100.0% chr18 + 35152141 35152161 21 browser details YourSeq 21 1363 1383 3000 100.0% chr17 + 86147246 86147266 21 browser details YourSeq 21 1266 1286 3000 100.0% chr14 + 69941916 69941936 21 browser details YourSeq 21 1361 1381 3000 100.0% chr11 + 60674611 60674631 21

Note: The 3000 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 3000 1 3000 3000 100.0% chr7 + 44854351 44857350 3000 browser details YourSeq 74 1791 2045 3000 96.3% chr5 - 145263902 145264442 541 browser details YourSeq 73 1312 1890 3000 76.5% chr11 - 58227897 58228400 504 browser details YourSeq 72 1674 1890 3000 91.9% chr11 + 110213639 110214008 370 browser details YourSeq 67 1790 1890 3000 91.4% chr1 + 74722951 74723556 606 browser details YourSeq 66 1794 1890 3000 90.3% chr5 - 134019398 134019497 100 browser details YourSeq 66 1791 1890 3000 84.6% chr11 - 70426341 70426442 102 browser details YourSeq 66 1792 1888 3000 94.6% chr1 - 176252178 176252277 100 browser details YourSeq 65 1759 1890 3000 97.2% chr12 + 108464955 108465138 184 browser details YourSeq 65 1776 1890 3000 93.4% chr10 + 91184018 91184132 115 browser details YourSeq 64 1804 1890 3000 95.8% chr4 + 148084907 148084996 90 browser details YourSeq 63 1791 1890 3000 94.3% chr4 - 132643683 132643787 105 browser details YourSeq 63 1791 1886 3000 93.2% chr19 - 47835273 47835373 101 browser details YourSeq 63 1804 1890 3000 95.7% chr1 - 191957761 191957850 90 browser details YourSeq 63 1792 2334 3000 68.0% chr8 + 108570501 108570643 143 browser details YourSeq 62 1791 1890 3000 97.0% chr6 - 142787161 142787262 102 browser details YourSeq 62 1792 1886 3000 93.1% chr2 + 91648451 91648548 98 browser details YourSeq 62 1791 1890 3000 95.6% chr19 + 22555989 22556091 103 browser details YourSeq 61 1793 1890 3000 93.1% chr16 - 32103127 32103224 98 browser details YourSeq 61 1792 1890 3000 95.6% chr15 - 99122640 99122741 102

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and 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

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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 (Comprehensive set... Il4i1-202 >protein coding Akt1s1-204 >protein coding Pnkp-203 >protein coding

Il4i1-201 >protein coding Akt1s1-201 >protein coding Pnkp-211 >protein coding Pnkp-215 >non stop decay

Il4i1-203 >retained intron Akt1s1-208 >protein coding Pnkp-201 >protein coding

Il4i1-204 >protein coding Akt1s1-210 >retained intron Pnkp-209 >retained intron

Akt1s1-209 >protein coding Pnkp-207 >nonsense mediated decay

Akt1s1-203 >protein coding Pnkp-205 >nonsense mediated decay

Page 6 oMf i r 7807-201 >miRNA Pnkp-202 >protein coding

Akt1s1-202 >protein coding Pnkp-204 >nonsense mediated decay

Akt1s1-207 >protein coding Pnkp-212 >nonsense mediated decay

Akt1s1-212 >protein coding Pnkp-214 >nonsense mediated decay

Akt1s1-206 >protein coding Pnkp-217 >protein coding

Akt1s1-211 >protein coding Pnkp-216 >nonsense mediated decay

Akt1s1-205 >protein coding Pnkp-210 >retained intron

Pnkp-206 >retained intron

Pnkp-213 >protein coding

Pnkp-208 >retained intron

Contigs AC158231.2 > Genes < Tbc1d17-201protein coding < Ptov1-205nonsense mediated decay (Comprehensive set...

< Tbc1d17-204retained intron < Ptov1-202retained intron

< Tbc1d17-206nonsense mediated decay < Ptov1-209retained intron

< Tbc1d17-202retained intron < Ptov1-204retained intron

< Tbc1d17-211nonsense mediated decay < Tbc1d17-205retained intron < Ptov1-211retained intron

< Tbc1d17-207retained intron < Tbc1d17-203retained intron < Ptov1-210retained intron

< Tbc1d17-208retained intron < Ptov1-201protein coding

< Tbc1d17-209protein coding < Ptov1-213protein coding

< Tbc1d17-212protein coding < Ptov1-206protein coding

< Tbc1d17-210protein coding < Ptov1-203protein coding

< Ptov1-208protein coding

< Ptov1-214lncRNA

Regulatory Build

44.84Mb 44.85Mb 44.86Mb Reverse strand 26.43 kb

Regulation Legend CTCF Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene 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

Il4i1-201 >protein coding Akt1s1-201 >protein coding Pnkp-211 >protein coding Pnkp-215 >non stop decay

Il4i1-203 >retained intron Akt1s1-208 >protein coding Pnkp-201 >protein coding

Il4i1-204 >protein coding Akt1s1-210 >retained intron Pnkp-209 >retained intron

Akt1s1-209 >protein coding Pnkp-207 >nonsense mediated decay

Akt1s1-203 >protein coding Pnkp-205 >nonsense mediated decay https://www.alphaknockout.com

Mir707-201 >miRNA Pnkp-202 >protein coding

Akt1s1-202 >protein coding Pnkp-204 >nonsense mediated decay

Akt1s1-207 >protein coding Pnkp-212 >nonsense mediated decay

Akt1s1-212 >protein coding Pnkp-214 >nonsense mediated decay

Akt1s1-206 >protein coding Pnkp-217 >protein coding

Akt1s1-211 >protein coding Pnkp-216 >nonsense mediated decay

Akt1s1-205 >protein coding Pnkp-210 >retained intron

Pnkp-206 >retained intron

Pnkp-213 >protein coding

Pnkp-208 >retained intron

Contigs AC158231.2 > Genes < Tbc1d17-201protein coding < Ptov1-205nonsense mediated decay (Comprehensive set...

< Tbc1d17-204retained intron < Ptov1-202retained intron

< Tbc1d17-206nonsense mediated decay < Ptov1-209retained intron

< Tbc1d17-202retained intron < Ptov1-204retained intron

< Tbc1d17-211nonsense mediated decay < Tbc1d17-205retained intron < Ptov1-211retained intron

< Tbc1d17-207retained intron < Tbc1d17-203retained intron < Ptov1-210retained intron

< Tbc1d17-208retained intron < Ptov1-201protein coding

< Tbc1d17-209protein coding < Ptov1-213protein coding

< Tbc1d17-212protein coding < Ptov1-206protein coding

< Tbc1d17-210protein coding < Ptov1-203protein coding

< Ptov1-208protein coding

< Ptov1-214lncRNA

Regulatory Build

44.84Mb 44.85Mb 44.86Mb Reverse strand 26.43 kb

Regulation Legend CTCF Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000107882

5.11 kb Forward strand

Akt1s1-203 >protein coding

ENSMUSP00000103... MobiDB lite Low complexity (Seg) Pfam Proline-rich AKT1 substrate 1 protein PANTHER Proline-rich AKT1 substrate 1 protein

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend stop gained inframe deletion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 283

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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