http://www.alphaknockout.com/ Mouse Atpif1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Atpif1 conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Atpif1 (NCBI Reference Sequence: NM_007512 ; Ensembl: ENSMUSG00000054428 ) is located on Mouse 4. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000067496). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Atpif1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-257O20 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 exhibit normal growth and breeding and are protected from TAC-pressure overload and isoproterenol infusion.

Exon 3 starts from about 56.6% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 2472 bp. The size of effective cKO region: ~1019 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Atpif1 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

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

Summary: Full Length(6769bp) | A(25.94% 1756) | C(23.22% 1572) | G(23.09% 1563) | T(27.74% 1878)

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% chr4 - 132531074 132534073 3000 browser details YourSeq 268 1590 2215 3000 89.7% chr15 - 81977100 81977935 836 browser details YourSeq 223 1613 2216 3000 83.7% chrX + 104582340 104582692 353 browser details YourSeq 218 1620 2165 3000 91.6% chr19 - 5163565 5164179 615 browser details YourSeq 217 1621 2167 3000 86.0% chr11 + 97360141 97360592 452 browser details YourSeq 216 1623 2222 3000 85.3% chr1 + 137052592 137052928 337 browser details YourSeq 214 1622 2225 3000 85.1% chr12 - 111578599 111578932 334 browser details YourSeq 214 1620 2227 3000 82.8% chr15 + 12252838 12253160 323 browser details YourSeq 213 1620 2226 3000 83.0% chr3 - 103162497 103162868 372 browser details YourSeq 213 1636 2216 3000 84.5% chr15 + 59220364 59220699 336 browser details YourSeq 211 1620 2215 3000 85.1% chr17 + 17429364 17429698 335 browser details YourSeq 209 1621 2217 3000 84.9% chr5 + 123331109 123331435 327 browser details YourSeq 209 1621 2214 3000 83.5% chr10 + 84985601 84985926 326 browser details YourSeq 207 1629 2219 3000 84.1% chr5 + 139467569 139467935 367 browser details YourSeq 205 1624 2214 3000 84.1% chr4 - 135639158 135639500 343 browser details YourSeq 204 1620 2206 3000 82.6% chr5 + 52935625 52935935 311 browser details YourSeq 204 1621 2217 3000 82.9% chr2 + 80675258 80675626 369 browser details YourSeq 204 1619 2217 3000 84.7% chr10 + 78411592 78411944 353 browser details YourSeq 203 1620 2218 3000 83.9% chr18 - 77860294 77860629 336 browser details YourSeq 200 1633 2227 3000 82.7% chr7 + 25871096 25871410 315

Note: The 3000 bp section upstream of Exon 3 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% chr4 - 132527555 132530554 3000 browser details YourSeq 209 2725 2982 3000 95.6% chr15 + 80168807 80169294 488 browser details YourSeq 204 2744 2982 3000 96.8% chr2 + 34472008 34472311 304 browser details YourSeq 203 2746 2982 3000 95.5% chr6 + 38528268 38528867 600 browser details YourSeq 202 2744 2984 3000 96.8% chrX + 159358046 159358647 602 browser details YourSeq 201 2752 2983 3000 95.9% chr7 - 122936011 122936623 613 browser details YourSeq 199 2724 2953 3000 94.0% chr5 + 147451730 147451954 225 browser details YourSeq 196 2745 2982 3000 91.5% chr2 + 91240238 91240469 232 browser details YourSeq 192 2730 2937 3000 98.0% chr3 - 131076886 131077099 214 browser details YourSeq 190 2726 2951 3000 91.7% chr15 - 8184716 8184923 208 browser details YourSeq 190 2726 2937 3000 93.0% chr3 + 122649319 122649518 200 browser details YourSeq 189 2730 2937 3000 94.0% chr1 + 170929608 170929807 200 browser details YourSeq 188 2725 2936 3000 96.6% chr15 - 100050089 100050309 221 browser details YourSeq 188 2725 2936 3000 93.3% chr10 + 121910593 121910786 194 browser details YourSeq 187 2744 2937 3000 96.4% chr13 - 55424200 55424389 190 browser details YourSeq 187 2724 2937 3000 97.0% chr7 + 49591198 49591483 286 browser details YourSeq 186 2726 2937 3000 93.5% chr7 - 116327736 116327936 201 browser details YourSeq 186 2733 2937 3000 97.0% chr13 - 97074355 97074561 207 browser details YourSeq 185 2729 2937 3000 93.1% chr3 - 145973794 145973995 202 browser details YourSeq 185 2746 2952 3000 93.9% chr5 + 38700931 38701129 199

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

Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Atpif1 ATPase inhibitory factor 1 [ Mus musculus (house mouse) ] Gene ID: 11983, updated on 17-Sep-2019

Gene summary

Official Symbol Atpif1 provided by MGI Official Full Name ATPase inhibitory factor 1 provided by MGI Primary source MGI:MGI:1196457 See related Ensembl:ENSMUSG00000054428 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as If1; Atpi; IF(1); ATP5IF1 Summary This gene encodes a member of the ATPase inhibitor family of proteins. This protein has been shown to negatively regulate Expression the ATP hydrolysis activity of the F1Fo-ATPase. Knockdown of this gene is associated with reduced heme synthesis in differentiating erythroid cells. Misregulation of this gene has been found to lead to increased aerobic glycolysis in mouse cancer cells, while high expression levels of this gene have been correlated with gastric and liver cancer severity in human patients. A pseudogene of this gene has been identified. [provided by RefSeq, Apr 2015] Orthologs Broad expression in liver E14 (RPKM 154.1), CNS E11.5 (RPKM 126.1) and 21 other tissues See more human all

Genomic context

Location: 4; 4 D2.3 See Atpif1 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (132530555..132535450, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (132086470..132089574, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 3 transcripts

Gene: Atpif1 ENSMUSG00000054428

Description ATPase inhibitory factor 1 [Source:MGI Symbol;Acc:MGI:1196457] Gene Synonyms If1 Location Chromosome 4: 132,530,555-132,533,659 reverse strand. GRCm38:CM000997.2 About this gene This gene has 3 transcripts (splice variants), 112 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Atpif1-201 ENSMUST00000067496.6 539 106aa ENSMUSP00000064282.6 Protein coding CCDS18727 O35143 TSL:1 GENCODE basic APPRIS P1

Atpif1-203 ENSMUST00000152993.7 462 74aa ENSMUSP00000133099.1 Protein coding - E9PV44 TSL:2 GENCODE basic

Atpif1-202 ENSMUST00000145795.1 1014 No protein - Retained intron - - TSL:2

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23.11 kb Forward strand 132.525Mb 132.530Mb 132.535Mb 132.540Mb Gm12999-201 >lncRNADnajc8-202 >protein coding (Comprehensive set...

Dnajc8-208 >nonsense mediated decay

Dnajc8-209 >retained intron

Dnajc8-210 >nonsense mediated decay

Dnajc8-206 >protein coding

Dnajc8-205 >protein coding

Dnajc8-201 >nonsense mediated decay

Dnajc8-207 >nonsense mediated decay

Dnajc8-211 >protein coding

Dnajc8-204 >retained intron

Contigs < AL805897.13 Genes < Atpif1-201protein coding (Comprehensive set...

< Atpif1-203protein coding

< Atpif1-202retained intron

Regulatory Build

132.525Mb 132.530Mb 132.535Mb 132.540Mb Reverse strand 23.11 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

Regulation Legend CTCF Promoter Promoter Flank

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Transcript: ENSMUST00000067496

< Atpif1-201protein coding

Reverse strand 3.10 kb

ENSMUSP00000064... Coiled-coils (Ncoils) Superfamily SSF64602 Mitochondrial ATPase inhibitor PANTHER PTHR23407

PTHR23407:SF6 Gene3D 1.20.5.500

All sequence SNPs/i... Sequence variants (dbSNP and all other sources) S R S Y K WR YK Y W

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 106

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

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