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Mouse Fgfbp1 Knockout Project (CRISPR/Cas9)

Objective: To create a Fgfbp1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fgfbp1 (NCBI Reference Sequence: NM_001271616 ; Ensembl: ENSMUSG00000048373 ) is located on Mouse 5. 3 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 3 (Transcript: ENSMUST00000199894). Exon 3 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 a knock-out allele exhibit abnormal neuromuscular synapse morphology and accelerates progression of ALS.

Exon 3 starts from about 0.13% of the coding region. Exon 3 covers 100.0% of the coding region. The size of effective KO region: ~751 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

Legends Exon of mouse Fgfbp1 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(25.4% 508) | C(23.9% 478) | T(28.4% 568) | G(22.3% 446)

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(29.0% 580) | C(18.3% 366) | T(28.45% 569) | G(24.25% 485)

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% chr5 - 43979949 43981948 2000 browser details YourSeq 50 263 460 2000 73.3% chr5 + 14314311 14314451 141 browser details YourSeq 44 274 443 2000 88.2% chr1 + 126836223 126836613 391 browser details YourSeq 41 263 460 2000 68.9% chr1 + 116692650 116692786 137 browser details YourSeq 35 406 460 2000 81.9% chr13 + 97290695 97290749 55 browser details YourSeq 33 397 439 2000 89.2% chr17 - 26658676 26658717 42 browser details YourSeq 33 220 280 2000 88.6% chr11 + 109390410 109390468 59 browser details YourSeq 32 397 438 2000 88.9% chr12 - 90043401 90043441 41 browser details YourSeq 32 404 438 2000 97.1% chr9 + 50758948 50758983 36 browser details YourSeq 32 397 440 2000 86.4% chr5 + 141919847 141919890 44 browser details YourSeq 32 409 443 2000 97.2% chr2 + 3273583 3273618 36 browser details YourSeq 32 362 434 2000 86.4% chr14 + 10551221 10551294 74 browser details YourSeq 30 427 460 2000 94.2% chr18 + 11724405 11724438 34 browser details YourSeq 29 402 434 2000 96.9% chr7 - 129185196 129185232 37 browser details YourSeq 29 405 436 2000 96.9% chr14 - 56584965 56584997 33 browser details YourSeq 29 405 443 2000 94.0% chr2 + 180941678 180941717 40 browser details YourSeq 28 423 460 2000 90.0% chr5 - 144383796 144383832 37 browser details YourSeq 28 408 439 2000 93.8% chr10 + 98776941 98776972 32 browser details YourSeq 27 411 441 2000 93.6% chr17 - 29133226 29133256 31 browser details YourSeq 27 405 445 2000 83.0% chr5 + 53192094 53192134 41

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% chr5 - 43977196 43979195 2000 browser details YourSeq 82 1680 1957 2000 89.4% chr2 + 172069873 172070213 341 browser details YourSeq 48 1130 1211 2000 79.3% chr14 - 75790081 75790162 82 browser details YourSeq 48 1161 1225 2000 87.7% chr11 - 88257374 88257865 492 browser details YourSeq 47 1132 1215 2000 84.1% chr18 - 38051919 38052005 87 browser details YourSeq 47 1127 1225 2000 73.8% chr10 + 89703712 89703810 99 browser details YourSeq 46 1729 1835 2000 81.1% chr11 - 114259409 114259517 109 browser details YourSeq 44 1720 1833 2000 96.0% chr8 - 9814680 9814797 118 browser details YourSeq 44 1718 1924 2000 94.0% chr13 + 95825203 95825480 278 browser details YourSeq 41 1124 1186 2000 82.6% chr18 - 38798515 38798577 63 browser details YourSeq 40 1824 1871 2000 91.7% chr3 - 92489991 92490038 48 browser details YourSeq 39 1718 1763 2000 88.4% chr7 + 140351247 140351290 44 browser details YourSeq 38 1245 1303 2000 92.9% chr17 - 43701120 43701178 59 browser details YourSeq 38 1718 1854 2000 78.1% chr5 + 37485173 37485299 127 browser details YourSeq 36 1132 1186 2000 95.0% chr18 + 10970778 10970832 55 browser details YourSeq 35 1131 1193 2000 77.8% chr15 - 83337274 83337336 63 browser details YourSeq 35 1902 1958 2000 89.8% chr12 + 99762601 99762656 56 browser details YourSeq 34 1727 1821 2000 86.9% chr8 - 8460966 8461058 93 browser details YourSeq 34 1124 1184 2000 92.5% chr11 + 97360520 97360581 62 browser details YourSeq 33 1723 1767 2000 76.4% chr2 + 61709663 61709700 38

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: Fgfbp1 fibroblast growth factor binding protein 1 [ Mus musculus (house mouse) ] Gene ID: 14181, updated on 12-Aug-2019

Gene summary

Official Symbol Fgfbp1 provided by MGI Official Full Name fibroblast growth factor binding protein 1 provided by MGI Primary source MGI:MGI:1096350 See related Ensembl:ENSMUSG00000048373 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 FGF-BP; FGF-BP1; FGFBP-1 Expression Biased expression in large intestine adult (RPKM 24.0), stomach adult (RPKM 14.7) and 11 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 B3 See Fgfbp1 in Genome Data Viewer Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (43978858..43981828, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (44370099..44373001, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 3 transcripts

Gene: Fgfbp1 ENSMUSG00000048373

Description fibroblast growth factor binding protein 1 [Source:MGI Symbol;Acc:MGI:1096350] Gene Synonyms FGF-BP Location Chromosome 5: 43,978,858-43,981,779 reverse strand. GRCm38:CM000998.2 About this gene This gene has 3 transcripts (splice variants), 202 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fgfbp1-202 ENSMUST00000199481.1 2191 251aa ENSMUSP00000143011.1 Protein coding CCDS19268 O70514 TSL:NA GENCODE basic APPRIS P1

Fgfbp1-203 ENSMUST00000199894.1 1228 251aa ENSMUSP00000142520.1 Protein coding CCDS19268 O70514 TSL:3 GENCODE basic APPRIS P1

Fgfbp1-201 ENSMUST00000061299.8 1161 251aa ENSMUSP00000056900.7 Protein coding CCDS19268 O70514 TSL:1 GENCODE basic APPRIS P1

22.92 kb Forward strand 43.97Mb 43.98Mb 43.99Mb Contigs AC164004.3 > (Comprehensive set... < Fgfbp1-203protein coding

< Fgfbp1-201protein coding

< Fgfbp1-202protein coding

Regulatory Build

43.97Mb 43.98Mb 43.99Mb Reverse strand 22.92 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

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Transcript: ENSMUST00000199894

< Fgfbp1-203protein coding

Reverse strand 2.92 kb

ENSMUSP00000142... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Pfam FGF binding 1

PANTHER FGF binding 1

PTHR15258:SF2

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

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 251

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

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