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Mouse Stac3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Stac3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Stac3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Stac3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Stac3 gene (NCBI Reference Sequence: NM_177707 ; Ensembl: ENSMUSG00000040287 ) is located on Mouse chromosome 10. 12 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000035839). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Stac3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-117H10 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: Homozygous inactivation of this gene leads to neonatal lethality, abnormal posture, thin diaphragm muscle, abnormal skeletal muscle morphology characterized by centralized nuclei and disorganized myofibrils, and impaired skeletal muscle contractility due to defective excitation-contraction coupling. Exon 6 starts from about 45.56% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 941 bp, and the size of intron 6 for 3'-loxP site insertion: 2270 bp. The size of effective cKO region: ~598 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 4 5 6 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Stac3 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7098bp) | A(25.39% 1802) | C(25.19% 1788) | T(23.13% 1642) | G(26.29% 1866) Note: The sequence of homologous arms and cKO region 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 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr10 + 127501609 127504608 3000 browser details YourSeq 106 707 876 3000 92.2% chr7 + 125771967 125772138 172 browser details YourSeq 90 702 875 3000 90.3% chr12 - 109852321 109852508 188 browser details YourSeq 90 707 880 3000 85.9% chr13 + 9583517 9583692 176 browser details YourSeq 89 707 876 3000 86.1% chr9 + 43083241 43083416 176 browser details YourSeq 84 708 873 3000 86.8% chr12 + 76355614 76355819 206 browser details YourSeq 82 707 881 3000 84.2% chr3 + 27802365 27802542 178 browser details YourSeq 80 708 876 3000 91.8% chr1 + 155807187 155807378 192 browser details YourSeq 78 710 880 3000 91.6% chr1 + 12490531 12490723 193 browser details YourSeq 75 711 882 3000 89.5% chr18 - 24868467 24868656 190 browser details YourSeq 75 699 875 3000 91.3% chr11 - 49968728 49968920 193 browser details YourSeq 74 707 876 3000 89.4% chr11 - 50045841 50046068 228 browser details YourSeq 73 707 869 3000 88.5% chr13 + 22225582 22225755 174 browser details YourSeq 72 710 882 3000 90.0% chr4 + 57078290 57078503 214 browser details YourSeq 72 707 880 3000 91.1% chr12 + 21467041 21467241 201 browser details YourSeq 71 707 876 3000 91.8% chr6 + 39076820 39077024 205 browser details YourSeq 71 707 886 3000 86.8% chr12 + 118185269 118185452 184 browser details YourSeq 69 707 874 3000 90.6% chr5 + 75631400 75631587 188 browser details YourSeq 68 708 877 3000 92.5% chr12 - 35183235 35183406 172 browser details YourSeq 68 756 876 3000 90.6% chr1 - 9500716 9500844 129 Note: The 3000 bp section upstream of Exon 6 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% chr10 + 127505207 127508206 3000 browser details YourSeq 237 937 1564 3000 87.1% chr13 - 24776695 24777129 435 browser details YourSeq 205 948 1566 3000 91.2% chr16 + 90302319 90313224 10906 browser details YourSeq 188 948 1564 3000 84.1% chr4 - 119579131 119579609 479 browser details YourSeq 187 961 1564 3000 91.3% chr9 + 56140376 56537447 397072 browser details YourSeq 170 982 1553 3000 83.1% chr11 + 19431744 19432148 405 browser details YourSeq 161 1380 1679 3000 87.1% chr2 + 154912581 154912852 272 browser details YourSeq 159 1356 1564 3000 87.7% chr11 + 50274487 50274675 189 browser details YourSeq 159 1386 1575 3000 93.1% chr10 + 40039514 40039704 191 browser details YourSeq 157 1374 1566 3000 92.6% chr15 - 10237685 10237882 198 browser details YourSeq 156 1365 1564 3000 92.0% chr1 - 33723129 33723343 215 browser details YourSeq 154 1380 1561 3000 93.2% chr9 - 66964360 66964540 181 browser details YourSeq 154 1379 1564 3000 91.9% chr3 - 20159042 20159228 187 browser details YourSeq 154 1386 1564 3000 93.3% chr7 + 117987220 117987400 181 browser details YourSeq 154 1394 1582 3000 91.4% chr11 + 104488129 104488318 190 browser details YourSeq 154 1378 1581 3000 88.4% chr11 + 87419591 87419790 200 browser details YourSeq 153 992 1552 3000 91.0% chr1 - 180881712 180882305 594 browser details YourSeq 152 1351 1564 3000 86.1% chr2 - 15046922 15047118 197 browser details YourSeq 152 1379 1564 3000 91.9% chr12 - 84237809 84238005 197 browser details YourSeq 151 960 1562 3000 83.2% chr9 - 111144973 111145365 393 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Stac3 SH3 and cysteine rich domain 3 [ Mus musculus (house mouse) ] Gene ID: 237611, updated on 24-Oct-2019 Gene summary Official Symbol Stac3 provided by MGI Official Full Name SH3 and cysteine rich domain 3 provided by MGI Primary source MGI:MGI:3606571 See related Ensembl:ENSMUSG00000040287 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 9830125E18 Expression Biased expression in mammary gland adult (RPKM 5.1), limb E14.5 (RPKM 4.7) and 11 other tissues See more Orthologs human all Genomic context Location: 10; 10 D3 See Stac3 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (127501617..127508823) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (126938773..126945871) Chromosome 10 - NC_000076.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Stac3 ENSMUSG00000040287 Description SH3 and cysteine rich domain 3 [Source:MGI Symbol;Acc:MGI:3606571] Location Chromosome 10: 127,501,686-127,508,823 forward strand. GRCm38:CM001003.2 About this gene This gene has 5 transcripts (splice variants), 210 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 38 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Stac3-201 ENSMUST00000035839.2 1790 360aa ENSMUSP00000048148.2 Protein coding CCDS24242 Q8BZ71 TSL:1 GENCODE basic APPRIS P1 Stac3-202 ENSMUST00000160019.7 1581 360aa ENSMUSP00000125124.1 Protein coding CCDS24242 Q8BZ71 TSL:1 GENCODE basic APPRIS P1 Stac3-203 ENSMUST00000160610.1 373 18aa ENSMUSP00000124638.1 Protein coding - E0CXX9 CDS 3' incomplete TSL:2 Stac3-205 ENSMUST00000162302.1 600 No protein - Retained intron - - TSL:3 Stac3-204 ENSMUST00000160760.1 560 No protein - Retained intron - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 27.14 kb Forward strand 127.495Mb 127.500Mb 127.505Mb 127.510Mb 127.515Mb Genes (Comprehensive set... R3hdm2-212 >protein coding Stac3-202 >protein coding Ndufa4l2-201 >protein coding R3hdm2-204 >protein coding Stac3-203 >protein coding Stac3-205 >retained intron Ndufa4l2-202 >lncRNA R3hdm2-202 >protein coding Stac3-201 >protein coding Ndufa4l2-203 >lncRNA R3hdm2-201 >protein coding Stac3-204 >retained intron R3hdm2-218 >protein coding R3hdm2-203 >protein coding R3hdm2-205 >protein coding R3hdm2-208 >nonsense mediated decay R3hdm2-210 >protein coding R3hdm2-216 >protein coding R3hdm2-214 >retained intron R3hdm2-213 >protein coding R3hdm2-219 >protein coding Contigs AC167719.2 > Genes < Shmt2-207retained intron (Comprehensive set... < Shmt2-203retained intron < Shmt2-206protein coding < Shmt2-201protein coding Regulatory Build 127.495Mb 127.500Mb 127.505Mb 127.510Mb 127.515Mb Reverse strand 27.14 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene
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