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Mouse Msto1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Msto1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Msto1 gene (NCBI Reference Sequence: NM_144898 ; Ensembl: ENSMUSG00000068922 ) is located on Mouse chromosome 3. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000126245). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Msto1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-255M8 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 3 starts from about 13.25% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 639 bp, and the size of intron 4 for 3'-loxP site insertion: 402 bp. The size of effective cKO region: ~676 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 4 5 6 7 8 9 10 11 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Msto1 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(7127bp) | A(25.23% 1798) | C(26.14% 1863) | T(23.46% 1672) | G(25.17% 1794)

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% chr3 - 88913178 88916177 3000 browser details YourSeq 359 463 973 3000 92.9% chr16 - 23088015 23088601 587 browser details YourSeq 287 509 974 3000 91.8% chr8 + 122563313 122563787 475 browser details YourSeq 272 630 985 3000 93.1% chr4 - 150273807 150274182 376 browser details YourSeq 270 635 965 3000 93.1% chr12 + 54678058 54678398 341 browser details YourSeq 269 640 985 3000 91.7% chr10 + 127364066 127364449 384 browser details YourSeq 264 635 971 3000 91.9% chr5 - 92115222 92115628 407 browser details YourSeq 252 659 969 3000 91.6% chr15 + 81459175 81459603 429 browser details YourSeq 252 452 958 3000 83.0% chr10 + 75260719 75261115 397 browser details YourSeq 249 630 983 3000 89.8% chr12 - 12952129 12952642 514 browser details YourSeq 239 451 969 3000 88.5% chr9 - 88517193 88517812 620 browser details YourSeq 231 451 759 3000 92.4% chr7 + 16935103 16935480 378 browser details YourSeq 231 451 905 3000 93.7% chr11 + 106928409 106929146 738 browser details YourSeq 227 459 758 3000 91.9% chr17 + 28884868 28885193 326 browser details YourSeq 226 640 971 3000 92.0% chr17 - 46392333 46392824 492 browser details YourSeq 225 636 965 3000 89.3% chr9 - 108283924 108284441 518 browser details YourSeq 225 649 1017 3000 90.4% chr15 - 73701951 73702540 590 browser details YourSeq 223 478 968 3000 91.6% chr10 - 127314761 127315274 514 browser details YourSeq 222 640 909 3000 94.1% chr10 - 121523826 121524591 766 browser details YourSeq 220 452 752 3000 92.1% chr2 + 112466491 112466810 320

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% chr3 - 88909502 88912501 3000 browser details YourSeq 53 8 111 3000 79.8% chr13 - 48213400 48213507 108 browser details YourSeq 45 20 79 3000 96.0% chr14 + 23994362 23994424 63 browser details YourSeq 43 20 143 3000 93.9% chr13 + 100607537 100607996 460 browser details YourSeq 41 33 91 3000 86.5% chr5 - 143934044 143934109 66 browser details YourSeq 39 10 80 3000 79.2% chr10 + 94155790 94155886 97 browser details YourSeq 38 35 94 3000 72.0% chr2 - 121010401 121010450 50 browser details YourSeq 37 5 52 3000 89.4% chr3 + 107823536 107823587 52 browser details YourSeq 37 11 179 3000 56.5% chr1 + 123003416 123003454 39 browser details YourSeq 36 28 78 3000 86.0% chr19 - 30182451 30182504 54 browser details YourSeq 35 29 129 3000 79.5% chr15 - 38698250 38698343 94 browser details YourSeq 35 30 110 3000 92.7% chr14 - 63905541 63905625 85 browser details YourSeq 34 40 80 3000 94.9% chr17 + 26048846 26048886 41 browser details YourSeq 34 28 78 3000 85.5% chr13 + 44949945 44949997 53 browser details YourSeq 34 32 80 3000 85.5% chr11 + 17547263 17547315 53 browser details YourSeq 33 41 82 3000 94.6% chr3 - 54473630 54473672 43 browser details YourSeq 33 20 143 3000 89.2% chr10 + 82755200 82755322 123 browser details YourSeq 31 28 82 3000 94.3% chr11 - 76690321 76690376 56 browser details YourSeq 30 32 80 3000 81.3% chr17 + 45988774 45988826 53 browser details YourSeq 28 20 77 3000 86.7% chr1 + 119468886 119468941 56

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Msto1 misato 1, mitochondrial distribution and morphology regulator [ Mus musculus (house mouse) ] Gene ID: 229524, updated on 12-Aug-2019

Gene summary

Official Symbol Msto1 provided by MGI Official Full Name misato 1, mitochondrial distribution and morphology regulator provided by MGI Primary source MGI:MGI:2385175 See related Ensembl:ENSMUSG00000068922 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 mst; BC008103 Expression Ubiquitous expression in testis adult (RPKM 97.5), ovary adult (RPKM 25.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 F1 See Msto1 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (88909616..88913950, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (88713538..88717872, complement)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 5 transcripts

Gene: Msto1 ENSMUSG00000068922

Description misato 1, mitochondrial distribution and morphology regulator [Source:MGI Symbol;Acc:MGI:2385175] Location Chromosome 3: 88,905,107-88,913,999 reverse strand. GRCm38:CM000996.2 About this gene This gene has 5 transcripts (splice variants), 189 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Msto1-202 ENSMUST00000126245.1 1875 556aa ENSMUSP00000115645.1 Protein coding CCDS17485 E9PUB7 TSL:1 GENCODE basic APPRIS P1

Msto1-201 ENSMUST00000107494.7 2093 586aa ENSMUSP00000103118.1 Protein coding - D3YX87 TSL:1 GENCODE basic

Msto1-205 ENSMUST00000147828.1 3891 No protein - Retained intron - - TSL:1

Msto1-204 ENSMUST00000137243.7 2636 No protein - Retained intron - - TSL:1

Msto1-203 ENSMUST00000128988.1 937 No protein - Retained intron - - TSL:3

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28.89 kb Forward strand

88.90Mb 88.91Mb 88.92Mb Genes Gon4l-203 >protein coding (Comprehensive set...

Gon4l-202 >protein coding

Gon4l-201 >protein coding

Gon4l-204 >retained intron n-R5s197-201 >rRNA

Contigs AC127377.4 > Genes (Comprehensive set... < Gm43713-201lncRNA < Msto1-201protein coding < Dap3-208retained intron

< Msto1-204retained intron < Dap3-201protein coding

< Msto1-202protein coding < Dap3-202protein coding

< Msto1-205retained intron < Dap3-212protein coding

< Msto1-203retained intron < Dap3-206protein coding

< Dap3-207protein coding

Regulatory Build

88.90Mb 88.91Mb 88.92Mb Reverse strand 28.89 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000126245

< Msto1-202protein coding

Reverse strand 4.38 kb

ENSMUSP00000115... Superfamily Tubulin/FtsZ, GTPase domain superfamily Pfam DML1/Misato, tubulin domain

Misato Segment II tubulin-like domain PANTHER PTHR13391

CDD cd06060

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

Variant Legend splice donor variant stop gained missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 556

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

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