https://www.alphaknockout.com

Mouse Fscn3 Knockout Project (CRISPR/Cas9)

Objective: To create a Fscn3 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fscn3 (NCBI Reference Sequence: NM_019569 ; Ensembl: ENSMUSG00000029707 ) is located on Mouse 6. 7 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 6 (Transcript: ENSMUST00000031719). Exon 1~6 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:

Exon 1 starts from about 0.07% of the coding region. Exon 1~6 covers 100.0% of the coding region. The size of effective KO region: ~8185 bp. The KO 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 6 7

Legends Exon of mouse Fscn3 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of start codon 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 2000 bp section downstream of stop codon 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(20.9% 418) | C(25.6% 512) | T(27.75% 555) | G(25.75% 515)

Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(26.95% 539) | C(21.5% 430) | T(27.65% 553) | G(23.9% 478)

Note: The 2000 bp section downstream of stop codon 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr6 + 28426017 28428016 2000 browser details YourSeq 53 421 482 2000 96.5% chrX + 135757125 135757331 207 browser details YourSeq 51 421 477 2000 96.4% chr1 - 171231950 171232213 264 browser details YourSeq 45 157 379 2000 88.7% chrX - 52770721 52770942 222 browser details YourSeq 41 425 482 2000 86.3% chr4 + 54919678 54919734 57 browser details YourSeq 38 425 514 2000 93.2% chr18 - 42829340 42829876 537 browser details YourSeq 38 421 474 2000 77.8% chr4 + 150547950 150547996 47 browser details YourSeq 37 421 469 2000 92.9% chr15 - 34911086 34911139 54 browser details YourSeq 37 421 482 2000 93.4% chr3 + 56322488 56322565 78 browser details YourSeq 35 424 463 2000 86.9% chr16 + 50502411 50502448 38 browser details YourSeq 32 428 467 2000 91.9% chr2 - 74798899 74798946 48 browser details YourSeq 30 421 452 2000 96.9% chr8 - 111551143 111551174 32 browser details YourSeq 30 421 456 2000 96.9% chr14 - 68226601 68226639 39 browser details YourSeq 29 1257 1300 2000 96.9% chr14 + 22552277 22552322 46 browser details YourSeq 29 448 482 2000 87.9% chr14 + 11096440 11096473 34 browser details YourSeq 29 421 451 2000 96.8% chr10 + 82338835 82338865 31 browser details YourSeq 28 421 450 2000 89.7% chr15 - 77134000 77134028 29 browser details YourSeq 26 423 451 2000 96.6% chr9 - 22554142 22554176 35 browser details YourSeq 25 1817 1841 2000 100.0% chr3 - 19000358 19000382 25 browser details YourSeq 25 421 445 2000 100.0% chr2 - 49852910 49852934 25

Note: The 2000 bp section upstream of start codon 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 2000 1 2000 2000 100.0% chr6 + 28436202 28438201 2000 browser details YourSeq 158 146 1374 2000 94.9% chr11 + 115454430 115751813 297384 browser details YourSeq 130 137 284 2000 92.2% chr11 + 88990348 88990489 142 browser details YourSeq 129 146 287 2000 92.5% chr12 + 40151280 40151412 133 browser details YourSeq 128 153 324 2000 87.9% chr12 - 4368402 4368542 141 browser details YourSeq 124 129 283 2000 90.4% chr11 + 88784256 88784401 146 browser details YourSeq 122 147 287 2000 90.8% chr11 + 85189941 85190070 130 browser details YourSeq 119 146 287 2000 88.8% chr10 + 83355555 83355687 133 browser details YourSeq 118 149 290 2000 91.8% chr1 - 152915405 152915544 140 browser details YourSeq 118 153 308 2000 92.1% chr15 + 36689649 36689997 349 browser details YourSeq 118 136 284 2000 91.3% chr11 + 59810464 59810611 148 browser details YourSeq 116 146 287 2000 90.9% chr14 + 102826355 102826496 142 browser details YourSeq 116 153 289 2000 89.2% chr14 + 79130633 79130761 129 browser details YourSeq 116 149 287 2000 89.1% chr11 + 101743488 101743615 128 browser details YourSeq 116 149 289 2000 88.0% chr11 + 6502012 6502144 133 browser details YourSeq 115 154 325 2000 94.6% chr14 + 65803441 65803664 224 browser details YourSeq 114 146 284 2000 87.7% chr13 + 8842115 8842244 130 browser details YourSeq 113 148 287 2000 87.6% chr13 + 100778127 100778255 129 browser details YourSeq 112 147 285 2000 87.0% chr11 - 119311782 119311911 130 browser details YourSeq 110 146 288 2000 85.1% chr2 + 130099311 130099444 134

Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Fscn3 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

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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 (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

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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.

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