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Mouse Igsf1 Knockout Project (CRISPR/Cas9)

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Mouse Igsf1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Igsf1 Knockout Project (CRISPR/Cas9) Objective: To create a Igsf1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Igsf1 gene (NCBI Reference Sequence: NM_177591 ; Ensembl: ENSMUSG00000031111 ) is located on Mouse chromosome X. 20 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 20 (Transcript: ENSMUST00000033442). Exon 2~9 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: Females homozygous for disruptions of this gene show no obvious phenotypic change. Hemizygous males show hypothyroidism and increased body weight. Exon 2 starts from the coding region. Exon 2~9 covers 40.22% of the coding region. The size of effective KO region: ~5326 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 6 7 8 9 20 Legends Exon of mouse Igsf1 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 1863 bp section downstream of Exon 9 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(29.5% 590) | C(19.95% 399) | T(26.9% 538) | G(23.65% 473) 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(1863bp) | A(27.75% 517) | C(23.4% 436) | T(28.45% 530) | G(20.4% 380) Note: The 1863 bp section downstream of Exon 9 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% chrX - 49795319 49797318 2000 browser details YourSeq 30 1540 1573 2000 94.2% chr10 - 120068490 120068523 34 browser details YourSeq 25 232 256 2000 100.0% chr4 + 86325363 86325387 25 browser details YourSeq 24 1545 1572 2000 92.9% chr4 - 128979760 128979787 28 browser details YourSeq 24 605 628 2000 100.0% chr16 + 50578265 50578288 24 browser details YourSeq 23 1547 1571 2000 100.0% chr13 - 70214926 70214953 28 browser details YourSeq 23 223 245 2000 100.0% chr2 + 96233501 96233523 23 browser details YourSeq 22 199 220 2000 100.0% chr9 - 39701050 39701071 22 browser details YourSeq 22 1426 1447 2000 100.0% chr15 - 59340966 59340987 22 browser details YourSeq 22 1417 1438 2000 100.0% chr8 + 22333400 22333421 22 browser details YourSeq 22 231 252 2000 100.0% chr5 + 62143490 62143511 22 browser details YourSeq 21 232 252 2000 100.0% chr5 - 149044781 149044801 21 browser details YourSeq 21 84 104 2000 100.0% chr4 + 59395876 59395896 21 browser details YourSeq 20 1547 1566 2000 100.0% chr1 - 25098503 25098522 20 browser details YourSeq 20 232 251 2000 100.0% chr1 + 92149582 92149601 20 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 1863 1 1863 1863 100.0% chrX - 49788219 49790081 1863 browser details YourSeq 25 1432 1461 1863 82.2% chr15 - 103013474 103013501 28 browser details YourSeq 25 912 942 1863 96.3% chr1 - 6183493 6183531 39 browser details YourSeq 24 1517 1540 1863 100.0% chr11 - 99038046 99038069 24 browser details YourSeq 23 1832 1854 1863 100.0% chr1 - 59135056 59135078 23 browser details YourSeq 22 1190 1214 1863 95.9% chr1 + 80998101 80998131 31 browser details YourSeq 20 938 959 1863 95.5% chr1 - 144704491 144704512 22 browser details YourSeq 20 576 595 1863 100.0% chr1 - 119424078 119424097 20 Note: The 1863 bp section downstream of Exon 9 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Igsf1 immunoglobulin superfamily, member 1 [ Mus musculus (house mouse) ] Gene ID: 209268, updated on 12-Aug-2019 Gene summary Official Symbol Igsf1 provided by MGI Official Full Name immunoglobulin superfamily, member 1 provided by MGI Primary source MGI:MGI:2147913 See related Ensembl:ENSMUSG00000031111 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 AI747649; mKIAA0364; 5330413N23; 5530402E03; InhBP/p120 Expression Biased expression in liver E14.5 (RPKM 9.1), liver E14 (RPKM 8.7) and 8 other tissues See more Orthologs human all Genomic context Location: X; X A5 See Igsf1 in Genome Data Viewer Exon count: 21 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (49782536..49797907, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (47135713..47150922, complement) Chromosome X - NC_000086.7 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Igsf1 ENSMUSG00000031111 Description immunoglobulin superfamily, member 1 [Source:MGI Symbol;Acc:MGI:2147913] Location Chromosome X: 49,782,536-49,797,749 reverse strand. GRCm38:CM001013.2 About this gene This gene has 5 transcripts (splice variants), 145 orthologues, 17 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Igsf1-201 ENSMUST00000033442.13 4421 1317aa ENSMUSP00000033442.7 Protein coding CCDS40964 Q7TQA1 TSL:1 GENCODE basic APPRIS P1 Igsf1-204 ENSMUST00000114893.7 2671 775aa ENSMUSP00000110543.1 Protein coding CCDS40965 Q7TQA1 TSL:1 GENCODE basic Igsf1-202 ENSMUST00000072037.12 2515 762aa ENSMUSP00000071919.6 Protein coding CCDS40966 Q7TQA1 TSL:1 GENCODE basic Igsf1-203 ENSMUST00000114891.1 1761 232aa ENSMUSP00000110541.1 Protein coding CCDS40967 Q7TQA1 TSL:1 GENCODE basic Igsf1-205 ENSMUST00000135492.1 417 No protein - lncRNA - - TSL:2 35.21 kb Forward strand 49.78Mb 49.79Mb 49.80Mb Contigs AL731789.9 > Genes (Comprehensive set... < Igsf1-204protein coding < Igsf1-203protein coding < Igsf1-201protein coding < Igsf1-202protein coding < Igsf1-205lncRNA Regulatory Build 49.78Mb 49.79Mb 49.80Mb Reverse strand 35.21 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000033442 < Igsf1-201protein coding Reverse strand 15.21 kb ENSMUSP00000033... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin subtype 2 Immunoglobulin subtype Pfam Immunoglobulin-like domain PROSITE profiles Immunoglobulin-like domain PANTHER PTHR11738:SF163 PTHR11738 Gene3D Immunoglobulin-like fold All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1317 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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