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Mouse Ndufaf2 Knockout Project (CRISPR/Cas9)

Objective: To create a Ndufaf2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ndufaf2 (NCBI Reference Sequence: NM_001127346.1 ; Ensembl: ENSMUSG00000068184 ) is located on Mouse 13. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000163558). Exon 4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit impaired complex I mitochondrial activity.

Exon 4 starts from about 51.39% of the coding region. Exon 4 covers 48.81% of the coding region. The size of effective KO region: ~246 bp. The KO 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 4

Legends Exon of mouse Ndufaf2 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 246 bp section of Exon 4 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 246 bp section of Exon 4 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(246bp) | A(38.21% 94) | C(21.54% 53) | T(19.11% 47) | G(21.14% 52)

Note: The 246 bp section of Exon 4 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(246bp) | A(37.8% 93) | C(21.54% 53) | T(18.7% 46) | G(21.95% 54)

Note: The 246 bp section of Exon 4 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 246 1 246 246 100.0% chr13 - 108052708 108052953 246 browser details YourSeq 54 77 240 246 85.4% chr17 - 92600125 92600385 261 browser details YourSeq 48 16 106 246 94.7% chr12 - 89978696 89978806 111 browser details YourSeq 47 13 65 246 98.1% chr3 + 35650788 35893678 242891 browser details YourSeq 29 31 68 246 94.0% chr1 - 122699327 122699385 59 browser details YourSeq 27 13 42 246 96.7% chr3 + 49438488 49438520 33 browser details YourSeq 26 13 42 246 96.5% chr3 + 49128668 49128700 33 browser details YourSeq 23 10 33 246 100.0% chr19 - 51227243 51227267 25 browser details YourSeq 23 14 41 246 96.0% chr3 + 45090199 45090229 31 browser details YourSeq 22 31 52 246 100.0% chr8 + 8508920 8508941 22 browser details YourSeq 20 222 241 246 100.0% chr15 - 91761159 91761178 20 browser details YourSeq 20 14 33 246 100.0% chr19 + 49973787 49973806 20 browser details YourSeq 20 31 52 246 95.5% chr14 + 32994675 32994696 22

Note: The 246 bp section 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 246 1 246 246 100.0% chr13 - 108052710 108052955 246 browser details YourSeq 91 79 242 246 78.8% chr17 - 92600125 92600385 261 browser details YourSeq 29 33 70 246 94.0% chr1 - 122699327 122699385 59 browser details YourSeq 28 18 47 246 100.0% chr12 - 89978755 89978806 52 browser details YourSeq 27 25 69 246 66.7% chr15 + 42193630 42193659 30 browser details YourSeq 23 12 35 246 100.0% chr19 - 51227243 51227267 25 browser details YourSeq 22 33 54 246 100.0% chr8 + 8508920 8508941 22

Note: The 246 bp section of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Ndufaf2 NADH:ubiquinone oxidoreductase complex assembly factor 2 [ Mus musculus (house mouse) ] Gene ID: 75597, updated on 26-Jun-2020

Gene summary

Official Symbol Ndufaf2 provided by MGI Official Full Name NADH:ubiquinone oxidoreductase complex assembly factor 2 provided by MGI Primary source MGI:MGI:1922847 See related Ensembl:ENSMUSG00000068184 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 C86051; mimitin; Ndufa12l; 1810058I14Rik Expression Broad expression in testis adult (RPKM 15.4), CNS E11.5 (RPKM 15.4) and 22 other tissues See more Orthologs human all

Genomic context

Location: 13; 13 D2.1 See Ndufaf2 in Genome Data Viewer Exon count: 6

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (108052589..108158652, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (108842783..108948819, complement)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 4 transcripts

Gene: Ndufaf2 ENSMUSG00000068184

Description NADH:ubiquinone oxidoreductase complex assembly factor 2 [Source:MGI Symbol;Acc:MGI:1922847] Gene Synonyms 1810058I14Rik, Ndufa12l, mimitin Location Chromosome 13: 108,002,715-108,158,623 reverse strand. GRCm38:CM001006.2 About this gene This gene has 4 transcripts (splice variants), 250 orthologues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ndufaf2- ENSMUST00000163558.2 678 168aa ENSMUSP00000130532.1 Protein coding CCDS49357 Q59J78 TSL:1 201 GENCODE basic APPRIS P1

Ndufaf2- ENSMUST00000223734.1 556 77aa ENSMUSP00000153480.1 Protein coding - A0A286YE33 GENCODE 202 basic

Ndufaf2- ENSMUST00000225830.1 250 83aa ENSMUSP00000153552.1 Protein coding - A0A286YE75 CDS 5' and 3' 204 incomplete

Ndufaf2- ENSMUST00000225702.1 2404 45aa ENSMUSP00000153468.1 Nonsense mediated - A0A286YDL9 - 203 decay

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175.91 kb Forward strand 108.00Mb 108.05Mb 108.10Mb 108.15Mb B230220B15Rik-202 >lincRNA Smim15-201 >protein coding Ercc8-205 >nonsense mediated decay (Comprehensive set...

B230220B15Rik-201 >lincRNA Smim15-202 >protein coding Ercc8-206 >nonsense mediated decay

Smim15-206 >protein coding Ercc8-210 >nonsense mediated decay

Smim15-205 >protein coding Ercc8-209 >nonsense mediated decay

Smim15-203 >protein coding Ercc8-204 >nonsense mediated decay

Smim15-204 >protein coding Ercc8-207 >nonsense mediated decay

Ercc8-203 >nonsense mediated decay

Ercc8-208 >retained intron

Ercc8-201 >protein coding

Ercc8-202 >protein coding

Contigs CT025566.7 > AC103379.8 > Genes (Comprehensive set... < Ndufaf2-203nonsense mediated decay

< Ndufaf2-201protein coding

< Ndufaf2-202protein coding

< Ndufaf2-204protein coding

Regulatory Build

108.00Mb 108.05Mb 108.10Mb 108.15Mb Reverse strand 175.91 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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

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Transcript: ENSMUST00000163558

< Ndufaf2-201protein coding

Reverse strand 106.03 kb

ENSMUSP00000130... MobiDB lite Low complexity (Seg) Pfam NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 PANTHER PTHR32470

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

Variant Legend

stop lost missense variant synonymous variant stop retained variant

Scale bar 0 20 40 60 80 100 120 140 168

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

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