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Mouse Bnc1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Bnc1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Bnc1 (NCBI Reference Sequence: NM_007562 ; Ensembl: ENSMUSG00000025105 ) is located on Mouse 7. 5 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 5 (Transcript: ENSMUST00000026096). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Bnc1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-129C5 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 null allele exhibit thinning and delayed wound healing of the corneal epithelium.

Exon 4 starts from about 14.58% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2163 bp, and the size of intron 4 for 3'-loxP site insertion: 4162 bp. The size of effective cKO region: ~2356 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 3 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Bnc1 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(8856bp) | A(26.68% 2363) | C(22.72% 2012) | T(28.86% 2556) | G(21.74% 1925)

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% chr7 - 81975296 81978295 3000 browser details YourSeq 138 1954 2183 3000 92.4% chr14 + 63867295 63867522 228 browser details YourSeq 110 1969 2178 3000 88.0% chr9 - 44042498 44042692 195 browser details YourSeq 94 2021 2173 3000 93.7% chr11 - 102700274 102700449 176 browser details YourSeq 70 2037 2173 3000 97.4% chr5 - 99358459 99359052 594 browser details YourSeq 64 2073 2190 3000 83.8% chr12 - 116256691 116256797 107 browser details YourSeq 62 2075 2178 3000 95.6% chr8 - 11152204 11152308 105 browser details YourSeq 62 2075 2183 3000 78.9% chr11 + 94769933 94837592 67660 browser details YourSeq 61 2071 2183 3000 91.8% chr4 - 141377967 141378081 115 browser details YourSeq 60 2071 2178 3000 77.8% chr6 + 51220441 51220548 108 browser details YourSeq 59 2132 2190 3000 100.0% chr13 - 43509965 43510023 59 browser details YourSeq 59 2081 2184 3000 76.5% chr17 + 25191740 25191807 68 browser details YourSeq 58 2075 2183 3000 81.9% chr7 - 35742845 35742940 96 browser details YourSeq 58 2084 2189 3000 81.2% chr7 + 127502146 127502243 98 browser details YourSeq 56 2117 2190 3000 82.1% chr18 - 12848961 12849028 68 browser details YourSeq 56 2078 2180 3000 81.9% chr11 - 51948334 51948427 94 browser details YourSeq 56 2132 2204 3000 89.1% chr19 + 41002920 41002999 80 browser details YourSeq 55 2134 2190 3000 98.3% chr14 + 106232598 106232654 57 browser details YourSeq 55 2132 2198 3000 91.1% chr10 + 76247367 76247433 67 browser details YourSeq 54 2132 2190 3000 96.7% chr17 - 47856564 47856840 277

Note: The 3000 bp section upstream of Exon 4 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% chr7 - 81969940 81972939 3000 browser details YourSeq 40 1589 1641 3000 88.7% chr10 + 23091424 23091474 51 browser details YourSeq 33 191 289 3000 89.2% chr10 + 85283236 85283332 97 browser details YourSeq 23 1989 2015 3000 84.7% chr4 + 119427491 119427516 26 browser details YourSeq 21 442 463 3000 100.0% chr11 - 77039054 77039076 23

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Bnc1 basonuclin 1 [ Mus musculus (house mouse) ] Gene ID: 12173, updated on 7-Oct-2019

Gene summary

Official Symbol Bnc1 provided by MGI Official Full Name basonuclin 1 provided by MGI Primary source MGI:MGI:1097164 See related Ensembl:ENSMUSG00000025105 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 Bnc; AI047752; AW546376 Expression Broad expression in ovary adult (RPKM 1.4), placenta adult (RPKM 1.2) and 18 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 D3 See Bnc1 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (81966657..81992299, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (89111548..89137185, complement)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 1 transcript

Gene: Bnc1 ENSMUSG00000025105

Description basonuclin 1 [Source:MGI Symbol;Acc:MGI:1097164] Location Chromosome 7: 81,966,653-81,992,307 reverse strand. GRCm38:CM001000.2 About this gene This gene has 1 transcript (splice variant), 194 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Bnc1-201 ENSMUST00000026096.9 4680 990aa ENSMUSP00000026096.7 Protein coding CCDS52285 F8VPY0 TSL:1 GENCODE basic APPRIS P1

45.66 kb Forward strand 81.96Mb 81.97Mb 81.98Mb 81.99Mb 82.00Mb Gm25907-201 >snoRNA 4833418N17Rik-201 >pseudogene (Comprehensive set...

Contigs < AC161216.4 Genes (Comprehensive set... < Bnc1-201protein coding

Regulatory Build

81.96Mb 81.97Mb 81.98Mb 81.99Mb 82.00Mb Reverse strand 45.66 kb

Regulation Legend CTCF Open Chromatin Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene RNA gene

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Transcript: ENSMUST00000026096

< Bnc1-201protein coding

Reverse strand 25.66 kb

ENSMUSP00000026... MobiDB lite Low complexity (Seg) SMART Zinc finger C2H2-type Pfam PF12874 PROSITE profiles Zinc finger C2H2-type PROSITE patterns Zinc finger C2H2-type PANTHER PTHR15021:SF1

Protein disconnected-like Gene3D 3.30.160.60

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

Variant Legend missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 990

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

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