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Mouse Dbn1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dbn1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dbn1 gene (NCBI Reference Sequence: NM_001177371 ; Ensembl: ENSMUSG00000034675 ) is located on Mouse chromosome 13. 15 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 15 (Transcript: ENSMUST00000021950). Exon 2~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dbn1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-97L6 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 a knock-out allele display impaired cued conditioning behavior. Mice homozygous for a different knock-out allele show altered neurotransmitter receptor levels in protein complexes, abnormal dendritic spine morphology, and impaired synaptic plasticity in the hippocampus.

Exon 2 starts from about 4.11% of the coding region. The knockout of Exon 2~8 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 4420 bp, and the size of intron 8 for 3'-loxP site insertion: 3512 bp. The size of effective cKO region: ~2594 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 2 3 4 5 6 7 8 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Dbn1 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(9094bp) | A(22.52% 2048) | C(24.88% 2263) | T(24.49% 2227) | G(28.11% 2556)

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.

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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% chr13 - 55483693 55486692 3000 browser details YourSeq 62 2113 2271 3000 84.6% chr10 - 84065868 84066018 151 browser details YourSeq 61 2111 2252 3000 92.9% chr16 - 95731758 95731901 144 browser details YourSeq 51 2116 2252 3000 75.0% chr11 - 84720782 84720885 104 browser details YourSeq 51 2105 2170 3000 91.4% chr10 - 41231056 41231120 65 browser details YourSeq 51 2101 2181 3000 82.2% chr10 + 32787778 32787848 71 browser details YourSeq 48 2115 2170 3000 94.6% chr11 - 90205366 90205422 57 browser details YourSeq 47 2115 2171 3000 94.4% chr15 - 61334585 61334643 59 browser details YourSeq 47 2086 2146 3000 96.3% chr1 + 150072343 150072617 275 browser details YourSeq 46 2113 2181 3000 76.5% chr12 + 110719738 110719789 52 browser details YourSeq 45 2119 2252 3000 94.2% chr16 - 28345074 28345256 183 browser details YourSeq 45 2115 2181 3000 79.6% chr1 - 157725233 157725283 51 browser details YourSeq 45 2110 2165 3000 96.0% chr16 + 68362568 68362632 65 browser details YourSeq 45 2105 2252 3000 65.4% chr1 + 151018496 151018545 50 browser details YourSeq 44 2118 2271 3000 65.4% chr14 + 12087859 12087917 59 browser details YourSeq 44 2113 2179 3000 75.6% chr13 + 87272949 87272998 50 browser details YourSeq 43 2115 2181 3000 77.6% chr12 - 31974630 31974683 54 browser details YourSeq 42 2115 2164 3000 88.9% chr14 - 28277837 28277883 47 browser details YourSeq 42 2111 2179 3000 82.9% chr12 + 112089418 112089502 85 browser details YourSeq 42 2115 2181 3000 77.3% chr10 + 104236504 104236548 45

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% chr13 - 55478099 55481098 3000 browser details YourSeq 150 532 909 3000 87.0% chr4 + 150699618 150699996 379 browser details YourSeq 120 532 913 3000 83.8% chr2 - 31695744 31696300 557 browser details YourSeq 104 705 910 3000 85.2% chr2 + 154626141 154626346 206 browser details YourSeq 98 536 884 3000 77.0% chr10 + 60020900 60021212 313 browser details YourSeq 92 745 907 3000 81.0% chr7 - 116516475 116516627 153 browser details YourSeq 91 792 1229 3000 71.8% chr6 + 39212232 39212394 163 browser details YourSeq 90 543 898 3000 85.8% chr10 + 43704053 43704505 453 browser details YourSeq 89 547 905 3000 90.1% chr15 - 101120186 101120701 516 browser details YourSeq 87 737 906 3000 85.3% chr13 - 117011629 117011801 173 browser details YourSeq 85 718 906 3000 81.4% chr7 + 24621334 24621521 188 browser details YourSeq 84 799 904 3000 89.7% chr5 + 150296436 150296541 106 browser details YourSeq 83 797 907 3000 87.4% chr1 - 180184485 180184595 111 browser details YourSeq 83 567 876 3000 85.0% chrX + 48638001 48638577 577 browser details YourSeq 83 802 914 3000 83.7% chr19 + 47185534 47185643 110 browser details YourSeq 82 547 858 3000 92.8% chr14 + 60349587 60350074 488 browser details YourSeq 81 793 905 3000 81.5% chr7 - 100291420 100291527 108 browser details YourSeq 81 792 904 3000 92.8% chr2 - 121090290 121090404 115 browser details YourSeq 80 801 910 3000 86.4% chr11 + 94195861 94195970 110 browser details YourSeq 79 715 908 3000 89.9% chr3 - 9741083 9741279 197

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Dbn1 drebrin 1 [ Mus musculus (house mouse) ] Gene ID: 56320, updated on 13-Aug-2019

Gene summary

Official Symbol Dbn1 provided by MGI Official Full Name drebrin 1 provided by MGI Primary source MGI:MGI:1931838 See related Ensembl:ENSMUSG00000034675 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 Expression Biased expression in whole brain E14.5 (RPKM 193.5), CNS E14 (RPKM 184.8) and 14 other tissues See more Orthologs human all

Genomic context

Location: 13 B1; 13 30.06 cM See Dbn1 in Genome Data Viewer Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (55473428..55488126, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (55574789..55589437, complement)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 9 transcripts

Gene: Dbn1 ENSMUSG00000034675

Description drebrin 1 [Source:MGI Symbol;Acc:MGI:1931838] Gene Synonyms drebrin A, drebrin E2 Location Chromosome 13: 55,473,429-55,488,111 reverse strand. GRCm38:CM001006.2 About this gene This gene has 9 transcripts (splice variants), 186 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 75 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dbn1- ENSMUST00000021950.14 3078 706aa ENSMUSP00000021950.8 Protein coding CCDS49274 Q9QXS6 TSL:1 201 GENCODE basic APPRIS ALT2

Dbn1- ENSMUST00000109923.8 2940 660aa ENSMUSP00000105549.2 Protein coding CCDS36677 Q3TRK3 TSL:1 203 Q9QXS6 GENCODE basic APPRIS P3

Dbn1- ENSMUST00000109921.8 2378 661aa ENSMUSP00000105547.2 Protein coding CCDS49273 A0A0R4J1E3 TSL:1 202 GENCODE basic APPRIS ALT2

Dbn1- ENSMUST00000139275.7 735 169aa ENSMUSP00000122574.1 Protein coding - F7CPL2 CDS 3' 207 incomplete TSL:3

Dbn1- ENSMUST00000135705.7 2119 No - Retained - - TSL:2 206 protein intron

Dbn1- ENSMUST00000127867.1 789 No - Retained - - TSL:5 205 protein intron

Dbn1- ENSMUST00000175813.1 732 No - Retained - - TSL:2 209 protein intron

Dbn1- ENSMUST00000139516.1 419 No - Retained - - TSL:3 208 protein intron

Dbn1- ENSMUST00000124480.7 573 No - lncRNA - - TSL:3 204 protein

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34.68 kb Forward strand

55.47Mb 55.48Mb 55.49Mb Genes Prr7-201 >protein coding (Comprehensive set...

Contigs < CT009762.8 Genes (Comprehensive set... < Dbn1-201protein coding < Pdlim7-211nonsense mediated decay

< Dbn1-203protein coding < Pdlim7-209retained intron

< Dbn1-202protein coding

< Dbn1-206retained intron

< Mir6944-201miRNA < Dbn1-205retained intron

< Dbn1-208retained intron

< Dbn1-209retained intron

< Dbn1-207protein coding

< Dbn1-204lncRNA

Regulatory Build

55.47Mb 55.48Mb 55.49Mb Reverse strand 34.68 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000021950

< Dbn1-201protein coding

Reverse strand 14.68 kb

ENSMUSP00000021... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF55753 SMART Actin-depolymerising factor homology domain Pfam Actin-depolymerising factor homology domain PROSITE profiles Actin-depolymerising factor homology domain PANTHER Drebrin

PTHR10829 Gene3D ADF-H/Gelsolin-like domain superfamily CDD cd11281

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 706

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

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