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Mouse Fscn3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Fscn3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Fscn3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Fscn3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fscn3 gene (NCBI Reference Sequence: NM_019569 ; Ensembl: ENSMUSG00000029707 ) is located on Mouse chromosome 6. 7 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 6 (Transcript: ENSMUST00000031719). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fscn3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-176I9 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 2 starts from about 9.71% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1817 bp, and the size of intron 2 for 3'-loxP site insertion: 839 bp. The size of effective cKO region: ~1197 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 5' gRNA region gRNA region 3' 1 2 3 7 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Fscn3 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. 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(7697bp) | A(24.31% 1871) | C(22.84% 1758) | T(28.73% 2211) | G(24.13% 1857) 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% chr6 + 28426728 28429727 3000 browser details YourSeq 155 2678 3000 3000 91.1% chr5 + 109668634 109669089 456 browser details YourSeq 154 2303 2844 3000 85.6% chr11 + 62028556 62029048 493 browser details YourSeq 148 2673 2844 3000 95.2% chr13 - 117349299 117349489 191 browser details YourSeq 143 2684 2852 3000 94.5% chr5 - 18464847 18465033 187 browser details YourSeq 137 2683 2847 3000 94.3% chr1 + 24015270 24015462 193 browser details YourSeq 136 2679 2844 3000 93.7% chr2 - 150821157 150821323 167 browser details YourSeq 134 2683 2841 3000 92.6% chrX - 20850680 20850836 157 browser details YourSeq 134 2683 2843 3000 90.7% chr15 - 80747559 80747713 155 browser details YourSeq 134 2679 2839 3000 93.6% chr14 - 17127327 17127493 167 browser details YourSeq 134 2678 2844 3000 94.8% chr6 + 5446841 5447034 194 browser details YourSeq 133 2678 2850 3000 87.9% chr16 + 4776531 4776697 167 browser details YourSeq 132 2683 2841 3000 91.2% chr1 + 162387881 162388036 156 browser details YourSeq 131 2683 2846 3000 94.0% chr6 - 37049196 37049361 166 browser details YourSeq 131 2674 2844 3000 87.5% chr19 + 26286879 26287044 166 browser details YourSeq 130 2683 2849 3000 87.4% chr7 + 122065037 122065195 159 browser details YourSeq 129 2683 2841 3000 95.2% chr9 - 60697063 60697227 165 browser details YourSeq 129 2683 2832 3000 94.5% chr7 - 116101440 116101589 150 browser details YourSeq 129 2678 2833 3000 91.0% chr2 - 144940118 144940270 153 browser details YourSeq 129 2683 2844 3000 93.8% chr2 - 107462151 107462312 162 Note: The 3000 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 3000 1 3000 3000 100.0% chr6 + 28430925 28433924 3000 browser details YourSeq 461 1080 1585 3000 95.9% chr1 + 127617625 127618136 512 browser details YourSeq 452 1080 1585 3000 95.1% chr5 - 68565105 68565619 515 browser details YourSeq 451 1080 1585 3000 95.4% chr15 - 42015656 42016160 505 browser details YourSeq 450 1080 1585 3000 95.0% chr14 - 30225856 30226362 507 browser details YourSeq 450 1080 1585 3000 94.9% chr15 + 71390932 71391447 516 browser details YourSeq 450 1080 1585 3000 95.1% chr12 + 52421935 52422441 507 browser details YourSeq 449 1080 1585 3000 95.6% chr7 - 111403301 111403809 509 browser details YourSeq 446 1080 1585 3000 95.6% chr18 + 86454647 86455155 509 browser details YourSeq 445 1080 1585 3000 95.1% chr8 + 82324211 82324714 504 browser details YourSeq 445 1081 1570 3000 96.1% chr2 + 158737697 158738189 493 browser details YourSeq 444 1080 1721 3000 94.4% chrX - 66510241 66511160 920 browser details YourSeq 444 1080 1578 3000 94.3% chr10 + 30696619 30697113 495 browser details YourSeq 443 1082 1585 3000 94.8% chr12 + 4755127 4755633 507 browser details YourSeq 442 1080 1579 3000 94.9% chr6 - 102848518 102849016 499 browser details YourSeq 442 1080 1585 3000 93.8% chr8 + 77456989 77457490 502 browser details YourSeq 441 1080 1585 3000 94.1% chr2 - 85555538 85556052 515 browser details YourSeq 440 1080 1585 3000 93.7% chrX - 51587905 51588406 502 browser details YourSeq 440 1080 1578 3000 95.2% chr3 - 124991269 124991774 506 browser details YourSeq 440 1080 1566 3000 95.4% chrX + 12957978 12958461 484 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Fscn3 fascin actin-bundling protein 3 [ Mus musculus (house mouse) ] Gene ID: 56223, updated on 10-Oct-2019 Gene summary Official Symbol Fscn3 provided by MGI Official Full Name fascin actin-bundling protein 3 provided by MGI Primary source MGI:MGI:1890386 See related Ensembl:ENSMUSG00000029707 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Expression Restricted expression toward testis adult (RPKM 130.6) See more Orthologs human all Genomic context Location: 6; 6 A3.3 See Fscn3 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (28427870..28438622) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (28377901..28388622) Chromosome 6 - NC_000072.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Fscn3 ENSMUSG00000029707 Description fascin actin-bundling protein 3 [Source:MGI Symbol;Acc:MGI:1890386] Location Chromosome 6: 28,427,789-28,438,622 forward strand. GRCm38:CM000999.2 About this gene This gene has 2 transcripts (splice variants), 107 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fscn3-201 ENSMUST00000031719.6 1902 498aa ENSMUSP00000031719.6 Protein coding CCDS39446 Q9QXW4 TSL:1 GENCODE basic APPRIS P1 Fscn3-202 ENSMUST00000147036.1 403 No protein - lncRNA - - TSL:2 30.83 kb Forward strand 28.42Mb 28.43Mb 28.44Mb Genes (Comprehensive set... Arf5-201 >protein codingFscn3-201 >protein coding Arf5-202 >protein coding Fscn3-202 >lncRNA Arf5-203 >retained intron Contigs AC068608.5 > Genes < Gcc1-202protein coding < Pax4-201protein coding (Comprehensive set... < Gcc1-201protein coding < Pax4-202protein coding < Gcc1-203protein coding < Pax4-203protein coding < Gcc1-204protein coding < Pax4-205protein coding < Pax4-206retained intron < Pax4-204retained intron Regulatory Build 28.42Mb 28.43Mb 28.44Mb Reverse strand 30.83 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000031719 10.83 kb Forward strand Fscn3-201 >protein coding ENSMUSP00000031... Superfamily Actin-crosslinking Pfam Fascin domain PIRSF Fascin, metazoans PANTHER Fascin-3 Fascin Gene3D 2.80.10.50 CDD Fascin domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 498 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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