https://www.alphaknockout.com

Mouse Fscn2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Fscn2 conditional 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 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fscn2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-296K20 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: 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 2 starts from about 56.03% 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: 4106 bp, and the size of intron 2 for 3'-loxP site insertion: 439 bp. The size of effective cKO region: ~626 bp. The cKO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 3 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fscn2 Homology arm cKO region loxP site

Page 2 of 8 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(7095bp) | A(22.61% 1604) | C(26.3% 1866) | T(22.51% 1597) | G(28.58% 2028)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

Page 3 of 8 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% chr11 + 120363392 120366391 3000 browser details YourSeq 218 778 2336 3000 94.0% chr18 - 31699893 31813741 113849 browser details YourSeq 150 804 970 3000 93.3% chr19 + 47335962 47336125 164 browser details YourSeq 150 716 968 3000 86.5% chr1 + 132261540 132261717 178 browser details YourSeq 147 799 969 3000 93.5% chr4 + 34694091 34694261 171 browser details YourSeq 147 805 970 3000 95.2% chr14 + 74730323 74730506 184 browser details YourSeq 146 801 970 3000 95.1% chr5 - 149723096 149723266 171 browser details YourSeq 143 811 975 3000 94.0% chr10 - 128738171 128738343 173 browser details YourSeq 141 808 965 3000 95.0% chr18 + 46491019 46491177 159 browser details YourSeq 140 809 969 3000 91.9% chr8 - 73276433 73276591 159 browser details YourSeq 140 801 969 3000 90.0% chr6 - 20678970 20679132 163 browser details YourSeq 140 778 952 3000 88.5% chr3 - 116371292 116371457 166 browser details YourSeq 138 809 970 3000 89.9% chr17 - 54283400 54283557 158 browser details YourSeq 137 809 970 3000 94.6% chr2 - 119522765 119522924 160 browser details YourSeq 133 810 970 3000 92.4% chr14 + 86479524 86479686 163 browser details YourSeq 130 801 973 3000 86.0% chr1 + 103787836 103787995 160 browser details YourSeq 127 809 945 3000 96.4% chr9 - 36841385 36841521 137 browser details YourSeq 127 804 952 3000 90.5% chr2 - 168736205 168736351 147 browser details YourSeq 127 808 952 3000 93.8% chr11 - 60898367 60898511 145 browser details YourSeq 126 798 953 3000 90.2% chr12 + 87259976 87260130 155

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% chr11 + 120367018 120370017 3000 browser details YourSeq 28 2517 2547 3000 96.7% chr13 - 93841269 93841307 39 browser details YourSeq 26 2630 2666 3000 85.8% chr4 + 17156987 17157021 35 browser details YourSeq 25 2383 2408 3000 100.0% chr1 - 15204375 15204405 31 browser details YourSeq 21 1863 1883 3000 100.0% chr12 - 100688686 100688706 21

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 https://www.alphaknockout.com

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

Page 5 of 8 https://www.alphaknockout.com

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

Page 6 of 8 https://www.alphaknockout.com

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

Page 7 of 8 https://www.alphaknockout.com

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.

Page 8 of 8