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Mouse Ndufb3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ndufb3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ndufb3 (NCBI Reference Sequence: NM_025597 ; Ensembl: ENSMUSG00000026032 ) is located on Mouse 1. 3 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000027193). 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 Ndufb3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-67I5 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:

Exon 2 starts from about 100% 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: 4419 bp, and the size of intron 2 for 3'-loxP site insertion: 4399 bp. The size of effective cKO region: ~658 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 3 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ndufb3 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(7158bp) | A(27.7% 1983) | C(19.1% 1367) | T(35.04% 2508) | G(18.16% 1300)

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% chr1 + 58587843 58590842 3000 browser details YourSeq 196 174 1090 3000 92.0% chr1 + 155407821 155524076 116256 browser details YourSeq 174 178 1073 3000 87.9% chr17 - 70732707 70842196 109490 browser details YourSeq 150 181 1082 3000 90.9% chr11 + 18010811 18127043 116233 browser details YourSeq 111 956 1090 3000 91.2% chr4 + 41139647 41139781 135 browser details YourSeq 109 954 1090 3000 89.8% chr9 - 114399985 114400121 137 browser details YourSeq 107 956 1090 3000 89.7% chr14 - 63592359 63592493 135 browser details YourSeq 106 956 1091 3000 89.0% chrX + 8269347 8269482 136 browser details YourSeq 105 956 1086 3000 90.1% chr17 - 87634310 87634440 131 browser details YourSeq 105 956 1086 3000 90.1% chr9 + 109938252 109938382 131 browser details YourSeq 105 957 1091 3000 88.9% chr2 + 130511142 130511276 135 browser details YourSeq 105 956 1090 3000 88.9% chr10 + 84616147 84616281 135 browser details YourSeq 105 956 1087 3000 90.1% chr1 + 118379472 118379604 133 browser details YourSeq 104 962 1091 3000 90.0% chr15 + 96793119 96793248 130 browser details YourSeq 103 962 1090 3000 90.0% chr4 - 123705151 123705279 129 browser details YourSeq 103 953 1090 3000 87.6% chr3 - 95876825 95876983 159 browser details YourSeq 103 956 1087 3000 89.4% chr14 - 122185755 122185890 136 browser details YourSeq 103 956 1086 3000 89.4% chr14 - 26509965 26510095 131 browser details YourSeq 103 956 1090 3000 88.2% chr15 + 61996687 61996821 135 browser details YourSeq 103 956 1091 3000 88.2% chr12 + 76848669 76849041 373

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% chr1 + 58591501 58594500 3000 browser details YourSeq 281 2503 2785 3000 99.0% chr10 - 66588787 66589068 282 browser details YourSeq 280 2505 2785 3000 100.0% chr18 + 17345959 17346248 290 browser details YourSeq 280 2504 2785 3000 99.7% chr15 + 52730822 52731103 282 browser details YourSeq 280 2505 2785 3000 100.0% chr10 + 119066246 119066558 313 browser details YourSeq 280 2504 2785 3000 100.0% chr1 + 105013715 105013998 284 browser details YourSeq 279 2506 2785 3000 100.0% chr15 - 18320727 18321030 304 browser details YourSeq 279 2506 2785 3000 100.0% chr13 - 6764247 6764552 306 browser details YourSeq 279 2503 2785 3000 99.7% chr10 + 14709279 14709578 300 browser details YourSeq 278 2508 2785 3000 100.0% chr12 - 64732742 64733019 278 browser details YourSeq 278 2508 2785 3000 100.0% chr10 - 29625847 29626124 278 browser details YourSeq 278 2503 2785 3000 99.3% chr1 - 146010942 146011297 356 browser details YourSeq 276 2504 2785 3000 99.3% chr16 - 40531757 40559118 27362 browser details YourSeq 276 2504 2785 3000 97.9% chr15 - 45932257 45932533 277 browser details YourSeq 276 2508 2785 3000 99.7% chr1 + 123318931 123319208 278 browser details YourSeq 275 2508 2785 3000 99.7% chr15 - 56891882 56892173 292 browser details YourSeq 275 2499 2785 3000 98.6% chr11 - 29444892 29445201 310 browser details YourSeq 275 2508 2785 3000 99.7% chr10 - 48058527 48058807 281 browser details YourSeq 275 2505 2785 3000 99.0% chr15 + 54204683 54204963 281 browser details YourSeq 275 2510 2785 3000 100.0% chr14 + 43662277 43662558 282

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

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Gene and information: Ndufb3 NADH:ubiquinone oxidoreductase subunit B3 [ Mus musculus (house mouse) ] Gene ID: 66495, updated on 12-Aug-2019

Gene summary

Official Symbol Ndufb3 provided by MGI Official Full Name NADH:ubiquinone oxidoreductase subunit B3 provided by MGI Primary source MGI:MGI:1913745 See related Ensembl:ENSMUSG00000026032 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 CI-B12; 2700033I16Rik Expression Ubiquitous expression in heart adult (RPKM 65.8), bladder adult (RPKM 47.1) and 26 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 C1.3 See Ndufb3 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (58586397..58595964)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (58643443..58652792)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 1 transcript

Gene: Ndufb3 ENSMUSG00000026032

Description NADH:ubiquinone oxidoreductase subunit B3 [Source:MGI Symbol;Acc:MGI:1913745] Gene Synonyms 2700033I16Rik Location Chromosome 1: 58,586,384-58,595,964 forward strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 206 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ndufb3-201 ENSMUST00000027193.8 763 104aa ENSMUSP00000027193.8 Protein coding CCDS14977 Q9CQZ6 TSL:1 GENCODE basic APPRIS P1

29.58 kb Forward strand

58.58Mb 58.59Mb 58.60Mb (Comprehensive set... Ndufb3-201 >protein coding

Contigs AC118698.7 > Genes < Fam126b-207protein coding (Comprehensive set...

< Fam126b-202protein coding

< Fam126b-201protein coding

< Fam126b-206protein coding

< Fam126b-205lncRNA

< Fam126b-204retained intron

< Fam126b-203retained intron

Regulatory Build

58.58Mb 58.59Mb 58.60Mb Reverse strand 29.58 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 processed transcript

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Transcript: ENSMUST00000027193

9.58 kb Forward strand

Ndufb3-201 >protein coding

ENSMUSP00000027... PDB-ENSP mappings Low complexity (Seg) Pfam NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3

PANTHER PTHR15082:SF2

NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3

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

Variant Legend

inframe insertion missense variant synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 104

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

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