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Mouse Fis1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Fis1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fis1 gene (NCBI Reference Sequence: NM_001163243 ; Ensembl: ENSMUSG00000019054 ) is located on Mouse chromosome 5. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000111094). 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 Fis1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-272B9 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 5.75% 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: 9408 bp, and the size of intron 2 for 3'-loxP site insertion: 1961 bp. The size of effective cKO region: ~633 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 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fis1 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(7133bp) | A(24.16% 1723) | C(24.98% 1782) | T(23.36% 1666) | G(27.51% 1962)

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% chr5 + 136959750 136962749 3000 browser details YourSeq 296 54 689 3000 95.8% chr5 + 136350476 136947726 597251 browser details YourSeq 192 100 489 3000 91.2% chr10 - 4244587 4245206 620 browser details YourSeq 191 51 290 3000 95.3% chr8 - 84206486 84206961 476 browser details YourSeq 186 54 291 3000 94.4% chr19 - 46051404 46051654 251 browser details YourSeq 182 54 292 3000 95.1% chr8 - 121557387 121557629 243 browser details YourSeq 182 86 292 3000 94.0% chr6 + 39415665 39415868 204 browser details YourSeq 182 101 609 3000 85.3% chr1 + 64761298 64761530 233 browser details YourSeq 181 97 294 3000 96.5% chr13 + 8599940 8874996 275057 browser details YourSeq 180 88 293 3000 94.7% chr11 + 20077643 20077860 218 browser details YourSeq 179 101 420 3000 88.2% chr4 - 109299558 109299810 253 browser details YourSeq 179 101 305 3000 95.5% chr12 + 24041011 24041221 211 browser details YourSeq 178 84 294 3000 95.9% chr17 + 6415445 6415655 211 browser details YourSeq 177 82 292 3000 92.9% chr4 - 132707832 132708038 207 browser details YourSeq 177 95 292 3000 95.5% chr19 - 45852638 45852843 206 browser details YourSeq 177 86 293 3000 93.9% chr15 - 31588319 31588524 206 browser details YourSeq 177 101 523 3000 87.7% chr11 - 76634990 76635355 366 browser details YourSeq 176 84 292 3000 92.3% chr9 - 75044461 75044663 203 browser details YourSeq 176 101 292 3000 96.4% chr9 - 43002835 43003031 197 browser details YourSeq 176 101 294 3000 95.9% chr2 - 172470648 172470849 202

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% chr5 + 136963383 136966382 3000 browser details YourSeq 95 1139 1285 3000 85.0% chr12 + 11324061 11324202 142 browser details YourSeq 94 1149 1285 3000 92.6% chr15 + 48958456 48958591 136 browser details YourSeq 92 1138 1280 3000 87.2% chr10 - 83665953 83666093 141 browser details YourSeq 91 1154 1349 3000 89.0% chr2 - 91724140 91724702 563 browser details YourSeq 88 1132 1285 3000 83.5% chr17 + 92070711 92070850 140 browser details YourSeq 87 1153 1281 3000 93.3% chr16 - 32122700 32123209 510 browser details YourSeq 87 1153 1285 3000 88.8% chr8 + 85371040 85371170 131 browser details YourSeq 86 1152 1280 3000 94.9% chrX + 73695626 73695755 130 browser details YourSeq 86 1141 1274 3000 87.9% chr2 + 91151814 91151945 132 browser details YourSeq 85 1153 1275 3000 84.5% chr1 - 185558781 185558899 119 browser details YourSeq 85 1180 1287 3000 93.0% chr1 + 156593950 156594061 112 browser details YourSeq 84 1139 1267 3000 86.3% chr10 + 128162171 128162295 125 browser details YourSeq 81 1155 1283 3000 89.8% chr4 + 139657170 139657297 128 browser details YourSeq 81 1149 1280 3000 88.2% chr15 + 99657396 99657522 127 browser details YourSeq 80 1194 1285 3000 94.6% chr7 + 129999385 129999480 96 browser details YourSeq 79 1153 1285 3000 85.6% chr14 + 74737335 74737463 129 browser details YourSeq 79 1153 1285 3000 85.0% chr12 + 75677320 75677494 175 browser details YourSeq 78 1188 1289 3000 89.9% chr2 - 25188507 25188619 113 browser details YourSeq 78 1140 1272 3000 86.3% chr11 - 3185843 3185974 132

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 protein information: Fis1 fission, mitochondrial 1 [ Mus musculus (house mouse) ] Gene ID: 66437, updated on 12-Aug-2019

Gene summary

Official Symbol Fis1 provided by MGI Official Full Name fission, mitochondrial 1 provided by MGI Primary source MGI:MGI:1913687 See related Ensembl:ENSMUSG00000019054 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 Ttc11; 2010003O14Rik Expression Ubiquitous expression in duodenum adult (RPKM 78.3), CNS E14 (RPKM 69.9) and 28 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 G2 See Fis1 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (136953275..136966234)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (137429145..137442104)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 6 transcripts

Gene: Fis1 ENSMUSG00000019054

Description fission, mitochondrial 1 [Source:MGI Symbol;Acc:MGI:1913687] Gene Synonyms 2010003O14Rik, Ttc11 Location Chromosome 5: 136,953,275-136,966,234 forward strand. GRCm38:CM000998.2 About this gene This gene has 6 transcripts (splice variants), 174 orthologues, 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

Fis1-202 ENSMUST00000111094.7 948 145aa ENSMUSP00000106723.1 Protein coding CCDS51669 G3X9U9 TSL:2 GENCODE basic

Fis1-201 ENSMUST00000019198.6 786 152aa ENSMUSP00000019198.6 Protein coding CCDS19757 Q9CQ92 TSL:1 GENCODE basic APPRIS P1

Fis1-203 ENSMUST00000111097.7 601 102aa ENSMUSP00000106726.1 Protein coding CCDS84977 D3YZ32 TSL:2 GENCODE basic

Fis1-205 ENSMUST00000147557.7 770 No protein - lncRNA - - TSL:5

Fis1-204 ENSMUST00000138610.7 723 No protein - lncRNA - - TSL:3

Fis1-206 ENSMUST00000152658.1 565 No protein - lncRNA - - TSL:2

32.96 kb Forward strand 136.95Mb 136.96Mb 136.97Mb Genes (Comprehensive set... Fis1-202 >protein coding Cldn15-202 >protein coding

Fis1-203 >protein coding Cldn15-201 >protein coding

Fis1-201 >protein coding Cldn15-203 >lncRNA

Fis1-204 >lncRNA

Fis1-205 >lncRNA

Fis1-206 >lncRNA

Contigs AC147987.5 > Regulatory Build

136.95Mb 136.96Mb 136.97Mb Reverse strand 32.96 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000111094

12.96 kb Forward strand

Fis1-202 >protein coding

ENSMUSP00000106... Transmembrane heli... Low complexity (Seg) Superfamily Tetratricopeptide-like helical domain superfamily Pfam Fis1, N-terminal tetratricopeptide repeat

Fis1, C-terminal tetratricopeptide repeat PIRSF Mitochondria fission 1 protein PANTHER Mitochondria fission 1 protein

PTHR13247:SF0 Gene3D Tetratricopeptide-like helical domain superfamily CDD Mitochondria fission protein Fis1, cytosolic domain

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

Variant Legend stop gained synonymous variant

Scale bar 0 20 40 60 80 100 120 145

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

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