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Mouse Ndufab1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ndufab1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ndufab1 gene (NCBI Reference Sequence: NM_028177 ; Ensembl: ENSMUSG00000030869 ) is located on Mouse chromosome 7. 5 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000033157). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ndufab1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-424B22 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 36.11% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 4923 bp, and the size of intron 3 for 3'-loxP site insertion: 1931 bp. The size of effective cKO region: ~3615 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ndufab1 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(10115bp) | A(26.36% 2666) | C(21.3% 2155) | T(30.13% 3048) | G(22.2% 2246)

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 - 122096982 122099981 3000 browser details YourSeq 327 216 1256 3000 94.6% chr6 + 113546950 113983199 436250 browser details YourSeq 318 209 1256 3000 94.3% chr12 + 59058204 59100774 42571 browser details YourSeq 213 222 862 3000 84.8% chrX + 162817654 162818047 394 browser details YourSeq 187 209 420 3000 94.3% chr7 + 100642174 100642389 216 browser details YourSeq 180 212 404 3000 95.3% chr5 - 34522538 34522728 191 browser details YourSeq 180 216 409 3000 95.4% chr2 + 32867029 32867221 193 browser details YourSeq 179 211 404 3000 96.4% chr9 - 115051505 115051699 195 browser details YourSeq 179 213 404 3000 96.9% chr10 - 77681745 77681943 199 browser details YourSeq 178 211 407 3000 95.9% chr19 + 38343796 38343998 203 browser details YourSeq 178 216 407 3000 96.4% chr1 + 145876172 145876363 192 browser details YourSeq 177 210 404 3000 94.4% chr10 + 40574390 40574583 194 browser details YourSeq 175 210 404 3000 95.9% chr15 - 75952223 75952424 202 browser details YourSeq 175 216 409 3000 95.9% chr13 - 99830670 99830906 237 browser details YourSeq 175 211 407 3000 95.0% chr8 + 95846393 95846719 327 browser details YourSeq 174 216 404 3000 96.3% chr18 - 7264098 7264292 195 browser details YourSeq 174 214 404 3000 93.6% chr11 + 96101129 96101315 187 browser details YourSeq 172 212 888 3000 86.2% chr5 - 110048037 110048689 653 browser details YourSeq 172 219 404 3000 96.3% chr2 - 84692975 84693160 186 browser details YourSeq 172 211 404 3000 96.3% chr18 - 60546535 60546728 194

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% chr7 - 122090367 122093366 3000 browser details YourSeq 328 2555 3000 3000 94.9% chr2 + 78832187 79394558 562372 browser details YourSeq 235 2713 3000 3000 92.0% chr13 - 6129928 6130214 287 browser details YourSeq 233 2708 3000 3000 92.1% chr5 - 22174804 22175072 269 browser details YourSeq 233 2574 3000 3000 89.1% chr6 + 57704469 57704740 272 browser details YourSeq 232 2552 3000 3000 89.2% chr16 + 73019148 73019425 278 browser details YourSeq 232 2713 3000 3000 90.7% chr14 + 22064314 22064588 275 browser details YourSeq 229 1681 2536 3000 90.6% chr7 - 139659544 139659777 234 browser details YourSeq 229 2550 3000 3000 93.9% chr19 + 27027609 27028063 455 browser details YourSeq 228 2729 3000 3000 91.1% chr14 - 118920178 118920437 260 browser details YourSeq 228 2572 3000 3000 88.4% chr1 - 183824664 183824923 260 browser details YourSeq 227 2712 3000 3000 92.9% chr1 - 113560690 113561280 591 browser details YourSeq 227 2729 3000 3000 92.3% chr4 + 48374588 48374854 267 browser details YourSeq 226 2728 3000 3000 94.1% chr16 - 39550234 39550505 272 browser details YourSeq 224 2712 3000 3000 92.1% chr1 - 18757727 18758004 278 browser details YourSeq 223 2743 3000 3000 93.8% chr2 - 78989884 78990150 267 browser details YourSeq 223 2748 3000 3000 95.6% chr1 - 55379427 55379691 265 browser details YourSeq 223 1681 2536 3000 89.7% chr7 + 80508162 80508392 231 browser details YourSeq 223 2713 3000 3000 90.6% chr5 + 96896354 96896638 285 browser details YourSeq 223 2729 3000 3000 94.9% chr16 + 31713527 31713896 370

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ndufab1 NADH:ubiquinone oxidoreductase subunit AB1 [ Mus musculus (house mouse) ] Gene ID: 70316, updated on 24-Oct-2019

Gene summary

Official Symbol Ndufab1 provided by MGI Official Full Name NADH:ubiquinone oxidoreductase subunit AB1 provided by MGI Primary source MGI:MGI:1917566 See related Ensembl:ENSMUSG00000030869 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 ACP; 8kDa; SDAP; CI-SDAP; 2210401F17Rik; 2310039H15Rik; 2610003B19Rik; 9130423F15Rik Expression Broad expression in adrenal adult (RPKM 768.6), duodenum adult (RPKM 586.0) and 16 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 F2 See Ndufab1 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (122086815..122101848, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (129231558..129245362, complement)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 10 transcripts

Gene: Ndufab1 ENSMUSG00000030869

Description NADH:ubiquinone oxidoreductase subunit AB1 [Source:MGI Symbol;Acc:MGI:1917566] Gene Synonyms 2210401F17Rik, 2310039H15Rik, 2610003B19Rik, 8kDa, 9130423F15Rik Location Chromosome 7: 122,085,403-122,101,886 reverse strand. GRCm38:CM001000.2 About this gene This gene has 10 transcripts (splice variants), 228 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ndufab1- ENSMUST00000033157.9 3479 156aa ENSMUSP00000033157.3 Protein coding CCDS21809 Q569N0 TSL:1 201 Q9CR21 GENCODE basic APPRIS P1

Ndufab1- ENSMUST00000123296.7 1416 156aa ENSMUSP00000116177.1 Protein coding CCDS21809 Q569N0 TSL:1 203 Q9CR21 GENCODE basic APPRIS P1

Ndufab1- ENSMUST00000106471.8 552 128aa ENSMUSP00000102079.2 Protein coding - F8WJ64 CDS 5' 202 incomplete TSL:3

Ndufab1- ENSMUST00000139456.1 452 127aa ENSMUSP00000114756.1 Protein coding - F6ZFT1 CDS 5' 206 incomplete TSL:2

Ndufab1- ENSMUST00000130857.1 1653 No - Retained - - TSL:1 204 protein intron

Ndufab1- ENSMUST00000153173.7 755 No - Retained - - TSL:2 210 protein intron

Ndufab1- ENSMUST00000146022.1 443 No - Retained - - TSL:2 208 protein intron

Ndufab1- ENSMUST00000130904.1 397 No - Retained - - TSL:2 205 protein intron

Ndufab1- ENSMUST00000146964.1 351 No - Retained - - TSL:3 209 protein intron

Ndufab1- ENSMUST00000145863.1 261 No - lncRNA - - TSL:5 207 protein

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36.48 kb Forward strand 122.08Mb 122.09Mb 122.10Mb 122.11Mb Genes Ubfd1-201 >protein coding Gm44986-201 >TEC (Comprehensive set...

Ubfd1-204 >retained intron

Ubfd1-205 >nonsense mediated decay

Contigs < AC124379.3 Genes < Ndufab1-201protein coding < Palb2-206nonsense mediated decay (Comprehensive set...

< Ndufab1-209retained intro

< Ndufab1-203protein coding < Palb2-208retained intron

< Ndufab1-204retained intron< Ndufab1-208retained intron < Palb2-202protein coding

< Ndufab1-202protein coding < Palb2-201protein coding

< Ndufab1-205retained intron < Palb2-204protein coding

< Ndufab1-206protein coding < Palb2-203protein coding

< Ndufab1-210retained intron

Regulatory Build

122.08Mb 122.09Mb 122.10Mb 122.11Mb Reverse strand 36.48 kb

Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000033157

< Ndufab1-201protein coding

Reverse strand 16.48 kb

ENSMUSP00000033... PDB-ENSP mappings Low complexity (Seg) TIGRFAM Acyl carrier protein (ACP)

Superfamily ACP-like superfamily Pfam Phosphopantetheine binding ACP domain PROSITE profiles Phosphopantetheine binding ACP domain PROSITE patterns Phosphopantetheine attachment site PANTHER PTHR20863

PTHR20863:SF5 HAMAP Acyl carrier protein (ACP) Gene3D ACP-like superfamily

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 20 40 60 80 100 120 156

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

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