https://www.alphaknockout.com

Mouse Fscn2 Knockout Project (CRISPR/Cas9)

Objective: To create a Fscn2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fscn2 (NCBI Reference Sequence: NM_172802 ; Ensembl: ENSMUSG00000025380 ) is located on Mouse 11. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000026445). Exon 1~5 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: Mice homozygous for disruptions in this gene display retinal generation with structural abnormalities of the outer segment and depressed rod and cone ERGs that worsen with age.

Exon 1 starts from about 0.07% of the coding region. Exon 1~5 covers 100.0% of the coding region. The size of effective KO region: ~6398 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

Legends Exon of mouse Fscn2 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(22.7% 454) | C(27.75% 555) | T(20.7% 414) | G(28.85% 577)

Note: The 2000 bp section upstream of start 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(22.4% 448) | C(26.95% 539) | T(19.85% 397) | G(30.8% 616)

Note: The 2000 bp section downstream of stop 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.

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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% chr11 + 120359710 120361709 2000 browser details YourSeq 23 1684 1708 2000 96.0% chr10 + 66056897 66056921 25 browser details YourSeq 22 6 27 2000 100.0% chr4 + 110185515 110185536 22 browser details YourSeq 22 730 751 2000 100.0% chr13 + 28799570 28799591 22 browser details YourSeq 20 898 917 2000 100.0% chr1 - 177518343 177518362 20

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% chr11 + 120368108 120370107 2000 browser details YourSeq 28 1427 1457 2000 96.7% chr13 - 93841269 93841307 39 browser details YourSeq 26 1540 1576 2000 85.8% chr4 + 17156987 17157021 35 browser details YourSeq 25 1293 1318 2000 100.0% chr1 - 15204375 15204405 31 browser details YourSeq 22 1443 1464 2000 100.0% chr7 + 4781120 4781141 22 browser details YourSeq 22 1807 1828 2000 100.0% chr16 + 96140200 96140221 22 browser details YourSeq 22 265 286 2000 100.0% chr12 + 26646375 26646396 22 browser details YourSeq 21 1893 1913 2000 100.0% chr7 - 19671741 19671761 21 browser details YourSeq 21 773 793 2000 100.0% chr12 - 100688686 100688706 21 browser details YourSeq 20 1362 1381 2000 100.0% chr1 + 80072517 80072536 20 browser details YourSeq 20 919 938 2000 100.0% chr1 + 36017980 36017999 20

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: Fscn2 actin-bundling protein 2 [ Mus musculus (house mouse) ] Gene ID: 238021, updated on 12-Aug-2019

Gene summary

Official Symbol Fscn2 provided by MGI Official Full Name fascin actin-bundling protein 2 provided by MGI Primary source MGI:MGI:2443337 See related Ensembl:ENSMUSG00000025380 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 Ahl8; A930022G03; C630046B20Rik Expression Low expression observed in reference dataset See more Orthologs human all

Genomic context

Location: 11; 11 E2 See Fscn2 in Genome Data Viewer Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (120360165..120368173)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (120222848..120229487)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 3 transcripts

Gene: Fscn2 ENSMUSG00000025380

Description fascin actin-bundling protein 2 [Source:MGI Symbol;Acc:MGI:2443337] Gene Synonyms C630046B20Rik, ahl8 Location Chromosome 11: 120,361,534-120,368,168 forward strand. GRCm38:CM001004.2 About this gene This gene has 3 transcripts (splice variants), 237 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 14 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fscn2-201 ENSMUST00000026445.2 1713 492aa ENSMUSP00000026445.2 Protein coding CCDS25731 Q32M02 TSL:1 GENCODE basic APPRIS P1

Fscn2-202 ENSMUST00000130476.1 669 No protein - lncRNA - - TSL:3

Fscn2-203 ENSMUST00000152556.1 591 No protein - lncRNA - - TSL:2

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26.64 kb Forward strand 120.355Mb 120.360Mb 120.365Mb 120.370Mb 120.375Mb (Comprehensive set... Fscn2-201 >protein coding

Fscn2-203 >lncRNA

Fscn2-202 >lncRNA

Contigs AL669855.20 > Genes < Faap100-204retained intron (Comprehensive set...

< Faap100-201protein coding

< Faap100-202retained intron < Faap100-205lncRNA

< Faap100-203lncRNA

< Faap100-208retained intron

< Faap100-207lncRNA

< Faap100-206lncRNA

Regulatory Build

120.355Mb 120.360Mb 120.365Mb 120.370Mb 120.375Mb Reverse strand 26.64 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000026445

6.63 kb Forward strand

Fscn2-201 >protein coding

ENSMUSP00000026... Superfamily Actin-crosslinking Pfam Fascin domain PIRSF Fascin, metazoans PANTHER Fascin-2

Fascin Gene3D 2.80.10.50 CDD Fascin domain

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 492

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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