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Mouse Flnb Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Flnb gene (NCBI Reference Sequence: NM_001081427 ; Ensembl: ENSMUSG00000025278 ) is located on Mouse chromosome 14. 46 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 46 (Transcript: ENSMUST00000052678). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Flnb gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-391P19 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: Mutations in this gene cause skeletal defects including runting, premature mineralization, and bone fusion. Nullizygous mice show a delay and reduction in long bone growth. Truncation mutations cause early fusion of spinal vertebrae due to enhanced chondrocyte hypertrophy and early differentiation.

Exon 5 starts from about 10.09% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 8090 bp, and the size of intron 6 for 3'-loxP site insertion: 1475 bp. The size of effective cKO region: ~1202 bp. The cKO 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 5 6 7 46 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Flnb Homology arm cKO region loxP site

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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(7702bp) | A(28.86% 2223) | C(21.98% 1693) | T(26.92% 2073) | G(22.24% 1713)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr14 + 7878334 7881333 3000 browser details YourSeq 84 2546 2679 3000 82.7% chr1 - 183746541 183746674 134 browser details YourSeq 80 2548 2675 3000 86.4% chr2 + 69207936 69208233 298 browser details YourSeq 75 2549 2695 3000 74.5% chr14 + 55474535 55474674 140 browser details YourSeq 74 778 2634 3000 91.2% chr10 - 116359736 116461057 101322 browser details YourSeq 69 2544 2683 3000 76.1% chr10 + 43567387 43567524 138 browser details YourSeq 65 2550 2680 3000 76.3% chr6 - 92391414 92391544 131 browser details YourSeq 63 2467 2635 3000 78.4% chrX - 99566876 99567371 496 browser details YourSeq 63 2575 2685 3000 76.4% chr15 - 96662907 96663016 110 browser details YourSeq 63 2550 2680 3000 82.8% chr13 - 54890095 54890223 129 browser details YourSeq 63 2565 2687 3000 75.7% chr11 - 120670593 120670715 123 browser details YourSeq 63 2567 2681 3000 75.5% chr11 - 107673645 107673758 114 browser details YourSeq 63 2546 2680 3000 77.7% chr11 - 100273215 100273347 133 browser details YourSeq 63 2566 2681 3000 88.4% chr15 + 75769799 75769913 115 browser details YourSeq 63 2546 2675 3000 78.8% chr12 + 84412844 84412971 128 browser details YourSeq 61 2552 2670 3000 72.9% chr2 - 157384246 157384358 113 browser details YourSeq 58 2573 2678 3000 77.4% chr5 - 150440541 150440646 106 browser details YourSeq 58 2572 2685 3000 80.0% chr9 + 108291429 108291540 112 browser details YourSeq 58 2572 2681 3000 76.4% chr5 + 114755934 114756043 110 browser details YourSeq 57 2546 2681 3000 71.2% chr17 - 44468533 44468668 136

Note: The 3000 bp section upstream of Exon 5 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% chr14 + 7882536 7885535 3000 browser details YourSeq 100 1688 1822 3000 87.1% chr11 - 80193688 80193821 134 browser details YourSeq 98 1678 1822 3000 84.9% chr9 + 107621814 107621958 145 browser details YourSeq 98 1691 1824 3000 87.2% chr1 + 156104655 156104788 134 browser details YourSeq 98 1697 1822 3000 89.6% chr1 + 85578652 85578778 127 browser details YourSeq 96 1689 1822 3000 84.9% chr4 + 145653771 145653903 133 browser details YourSeq 93 1703 1822 3000 86.5% chr1 - 85115740 85115857 118 browser details YourSeq 93 1711 1875 3000 84.3% chr1 + 74354088 74354239 152 browser details YourSeq 92 1692 1822 3000 83.0% chr18 - 57703007 57703135 129 browser details YourSeq 90 1724 1870 3000 86.4% chr10 - 43867404 43867559 156 browser details YourSeq 88 1690 1822 3000 88.6% chr19 - 10172426 10172580 155 browser details YourSeq 86 1695 1828 3000 85.9% chr5 - 114898175 114898367 193 browser details YourSeq 86 1690 1814 3000 85.3% chr10 - 76876642 76876771 130 browser details YourSeq 81 1712 1828 3000 88.0% chr1 - 171302799 171302915 117 browser details YourSeq 81 1722 1822 3000 90.1% chr17 + 81994094 81994194 101 browser details YourSeq 80 1685 1822 3000 85.1% chr1 - 170152076 170152212 137 browser details YourSeq 80 1735 1828 3000 92.6% chr5 + 65727036 65727129 94 browser details YourSeq 79 1691 1804 3000 88.5% chr8 + 128648668 128648992 325 browser details YourSeq 79 1711 1822 3000 92.6% chr7 + 125498525 125615550 117026 browser details YourSeq 78 1676 1822 3000 91.5% chr17 - 35804916 35805234 319

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Flnb filamin, beta [ Mus musculus (house mouse) ] Gene ID: 286940, updated on 12-Aug-2019

Gene summary

Official Symbol Flnb provided by MGI Official Full Name filamin, beta provided by MGI Primary source MGI:MGI:2446089 See related Ensembl:ENSMUSG00000025278 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 Fln-b; AL024016 Expression Ubiquitous expression in colon adult (RPKM 35.6), placenta adult (RPKM 32.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 A1 See Flnb in Genome Data Viewer

Exon count: 46

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (7817921..7951588)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (8650471..8784102)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 2 transcripts

Gene: Flnb ENSMUSG00000025278

Description filamin, beta [Source:MGI Symbol;Acc:MGI:2446089] Location Chromosome 14: 7,817,957-7,951,588 forward strand. GRCm38:CM001007.2 About this gene This gene has 2 transcripts (splice variants), 215 orthologues, 36 paralogues, is a member of 1 Ensembl protein family and is associated with 83 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Flnb-201 ENSMUST00000052678.8 9100 2602aa ENSMUSP00000052020.8 Protein coding CCDS70540 Q80X90 TSL:5 GENCODE basic APPRIS P1

Flnb-202 ENSMUST00000228206.1 111 No protein - lncRNA - - -

153.63 kb Forward strand 7.82Mb 7.84Mb 7.86Mb 7.88Mb 7.90Mb 7.92Mb 7.94Mb 7.96Mb Genes (Comprehensive set... Flnb-201 >protein coding

Flnb-202 >lncRNA Gm45521-201 >protein coding

Contigs AC129222.4 > < AC140322.4 Regulatory Build

7.82Mb 7.84Mb 7.86Mb 7.88Mb 7.90Mb 7.92Mb 7.94Mb 7.96Mb Reverse strand 153.63 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000052678

133.63 kb Forward strand

Flnb-201 >protein coding

ENSMUSP00000052... Low complexity (Seg) Superfamily Immunoglobulin E-set

CH domain superfamily SMART Calponin homology domain

Filamin/ABP280 repeat Pfam Calponin homology domain

Filamin/ABP280 repeat-like PROSITE profiles Filamin/ABP280 repeat-like

Calponin homology domain PROSITE patterns Actinin-type actin-binding domain, conserved site

Actinin-type actin-binding domain, conserved site PANTHER Filamin-B

PTHR38537 Gene3D CH domain superfamily

Immunoglobulin-like fold CDD Calponin homology domain

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

Variant Legend stop gained missense variant splice region variant synonymous variant

Scale bar 0 400 800 1200 1600 2000 2602

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

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