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Mouse Serpinb7 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Serpinb7 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Serpinb7 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Serpinb7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Serpinb7 gene (NCBI Reference Sequence: NM_027548 ; Ensembl: ENSMUSG00000067001 ) is located on Mouse chromosome 1. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000086690). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Serpinb7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-124P23 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: Exon 5 starts from about 29.56% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 10548 bp, and the size of intron 5 for 3'-loxP site insertion: 1998 bp. The size of effective cKO region: ~618 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 5 6 8 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Serpinb7 Homology arm cKO region loxP site Page 2 of 7 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. 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(7118bp) | A(30.84% 2195) | C(18.05% 1285) | T(32.85% 2338) | G(18.26% 1300) 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 7 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% chr1 + 107442743 107445742 3000 browser details YourSeq 422 1885 2416 3000 89.9% chr5 + 124948977 124949524 548 browser details YourSeq 231 35 1009 3000 85.2% chr1 - 16882061 16883854 1794 browser details YourSeq 227 647 1494 3000 89.4% chr9 + 124111250 124112106 857 browser details YourSeq 219 792 1375 3000 87.3% chr1 + 55259873 55260462 590 browser details YourSeq 196 825 1547 3000 86.4% chr12 - 55748594 55749317 724 browser details YourSeq 190 383 1307 3000 85.6% chr17 - 15456768 15457723 956 browser details YourSeq 176 788 1275 3000 84.4% chr8 + 23307559 23308048 490 browser details YourSeq 176 667 1139 3000 85.2% chr12 + 52679854 52680333 480 browser details YourSeq 170 604 1541 3000 80.2% chr1 + 47517034 47517832 799 browser details YourSeq 164 930 1689 3000 76.0% chr9 + 62251075 62251696 622 browser details YourSeq 163 567 1556 3000 82.2% chr13 - 114801819 114802582 764 browser details YourSeq 162 1060 1494 3000 74.3% chr6 + 31001595 31001979 385 browser details YourSeq 162 929 1305 3000 89.1% chr11 + 77284802 77285292 491 browser details YourSeq 159 1086 1420 3000 81.2% chr16 + 33120096 33120824 729 browser details YourSeq 158 928 1375 3000 83.5% chr13 - 14391322 14391746 425 browser details YourSeq 157 829 1377 3000 84.1% chr4 + 115522932 115608867 85936 browser details YourSeq 155 918 1418 3000 86.5% chr12 - 109295410 109367038 71629 browser details YourSeq 154 312 879 3000 87.1% chrX - 17538739 17539370 632 browser details YourSeq 151 794 1547 3000 87.2% chr19 + 36282004 36282809 806 Note: The 3000 bp section upstream of Exon 5 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% chr1 + 107446361 107449360 3000 browser details YourSeq 30 785 848 3000 97.0% chr14 - 49895606 49895671 66 browser details YourSeq 28 1798 1829 3000 93.8% chr1 + 107523115 107523146 32 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Serpinb7 serine (or cysteine) peptidase inhibitor, clade B, member 7 [ Mus musculus (house mouse) ] Gene ID: 116872, updated on 12-Aug-2019 Gene summary Official Symbol Serpinb7 provided by MGI Official Full Name serine (or cysteine) peptidase inhibitor, clade B, member 7 provided by MGI Primary source MGI:MGI:2151053 See related Ensembl:ENSMUSG00000067001 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 megsin; ovalbumin; 4631416M05Rik Expression Low expression observed in reference dataset See more Orthologs human all Genomic context Location: 1; 1 E2.1 See Serpinb7 in Genome Data Viewer Exon count: 8 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (107422689..107452689) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (109319266..109349266) Chromosome 1 - NC_000067.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Serpinb7 ENSMUSG00000067001 Description serine (or cysteine) peptidase inhibitor, clade B, member 7 [Source:MGI Symbol;Acc:MGI:2151053] Gene Synonyms 4631416M05Rik, megsin, ovalbumin Location Chromosome 1: 107,399,655-107,452,689 forward strand. GRCm38:CM000994.2 About this gene This gene has 2 transcripts (splice variants), 98 orthologues, 63 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Serpinb7-201 ENSMUST00000086690.5 1887 380aa ENSMUSP00000083896.4 Protein coding CCDS35686 Q9D695 TSL:1 GENCODE basic APPRIS P1 Serpinb7-202 ENSMUST00000154538.7 380 97aa ENSMUSP00000119217.1 Protein coding - D3Z2N5 CDS 3' incomplete TSL:2 73.03 kb Forward strand 107.40Mb 107.42Mb 107.44Mb 107.46Mb Genes (Comprehensive set... Serpinb7-202 >protein coding Serpinb7-201 >protein coding Contigs < AC157660.2 AC129295.4 > < AC148982.4 Genes < Gm24553-201snoRNA (Comprehensive set... Regulatory Build 107.40Mb 107.42Mb 107.44Mb 107.46Mb Reverse strand 73.03 kb Regulation Legend CTCF Enhancer Open Chromatin Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000086690 30.00 kb Forward strand Serpinb7-201 >protein coding ENSMUSP00000083... Low complexity (Seg) Superfamily Serpin superfamily SMART Serpin domain Pfam Serpin domain PROSITE patterns Serpin, conserved site PANTHER Serpin family PTHR11461:SF56 Gene3D Serpin superfamily, domain 2 Serpin superfamily, domain 1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 380 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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