https://www.alphaknockout.com

Mouse Mtrr Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Mtrr gene (NCBI Reference Sequence: NM_172480 ; Ensembl: ENSMUSG00000034617 ) is located on Mouse chromosome 13. 15 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000045827). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mtrr gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-85H21 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 homozygous for a hypomorphic gene trap allele are viable and display a male-specific reduction in postnatal weight gain as well as hyperhomocysteinemia, hypomethionemia, increased tissue methyltetrahydrofolate, and AdoMet/AdoHcy ratios that range from high to slightly below normal.

Exon 5 starts from about 19.25% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2197 bp, and the size of intron 5 for 3'-loxP site insertion: 2317 bp. The size of effective cKO region: ~873 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 4 5 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mtrr 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(7373bp) | A(22.92% 1690) | C(22.32% 1646) | T(30.14% 2222) | G(24.62% 1815)

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% chr13 - 68575641 68578640 3000 browser details YourSeq 32 1075 1126 3000 81.1% chr2 + 91303782 91303830 49

Note: The 3000 bp section upstream of Exon 5 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% chr13 - 68571768 68574767 3000 browser details YourSeq 479 176 1148 3000 86.4% chr1 - 10348397 10349182 786 browser details YourSeq 447 165 1147 3000 86.6% chr8 - 84298953 84299705 753 browser details YourSeq 405 173 1122 3000 85.7% chr4 + 14966931 14967633 703 browser details YourSeq 398 174 1147 3000 86.3% chr12 - 71730376 71731129 754 browser details YourSeq 397 395 1128 3000 85.8% chr2 + 25711412 25712087 676 browser details YourSeq 372 173 1022 3000 85.5% chr3 - 16101594 16412530 310937 browser details YourSeq 370 483 1147 3000 85.8% chr15 - 35158340 35158990 651 browser details YourSeq 366 395 1148 3000 87.2% chr16 + 32534043 32534788 746 browser details YourSeq 347 173 1147 3000 82.8% chr18 - 88911412 88912173 762 browser details YourSeq 343 430 1147 3000 85.8% chr4 + 43799194 43799885 692 browser details YourSeq 341 171 1080 3000 82.7% chr18 + 6882927 6883617 691 browser details YourSeq 334 165 977 3000 84.7% chr2 - 25749256 25749871 616 browser details YourSeq 306 395 1086 3000 82.2% chr2 - 149318771 149319432 662 browser details YourSeq 304 173 1112 3000 84.4% chr17 - 74922687 74923458 772 browser details YourSeq 297 395 1112 3000 85.2% chr11 - 73383208 73383912 705 browser details YourSeq 291 395 1147 3000 84.7% chr2 - 18475031 18475765 735 browser details YourSeq 283 483 1129 3000 89.5% chr15 - 34285008 34285663 656 browser details YourSeq 278 173 970 3000 83.1% chr10 - 41260213 41260773 561 browser details YourSeq 272 395 1147 3000 85.8% chr9 - 22544553 22545320 768

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Mtrr 5-methyltetrahydrofolate-homocysteine methyltransferase reductase [ Mus musculus (house mouse) ] Gene ID: 210009, updated on 12-Aug-2019

Gene summary

Official Symbol Mtrr provided by MGI Official Full Name 5-methyltetrahydrofolate-homocysteine methyltransferase reductase provided by MGI Primary source MGI:MGI:1891037 See related Ensembl:ENSMUSG00000034617 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 MSR; 4732420G08 Summary Methionine is an essential amino acid required for protein synthesis and one-carbon metabolism. Its synthesis is catalyzed Expression by the enzyme methionine synthase. Methionine synthase eventually becomes inactive due to the oxidation of its cob(I)alamin cofactor. The protein encoded by this gene regenerates a functional methionine synthase via reductive methylation. It is a member of the ferredoxin-NADP(+) reductase (FNR) family of electron transferases. Mutations in a similar gene in human have been associated with cblE complementation type homocystinuria-megaloblastic anemia and susceptibility to folate-sensitive neural tube defects. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, May 2015] Orthologs Ubiquitous expression in limb E14.5 (RPKM 4.4), placenta adult (RPKM 3.4) and 28 other tissues See more human all

Genomic context

Location: 13 B3; 13 35.54 cM See Mtrr in Genome Data Viewer Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (68560780..68582169, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (68699657..68720998, complement)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 9 transcripts

Gene: Mtrr ENSMUSG00000034617

Description 5-methyltetrahydrofolate-homocysteine methyltransferase reductase [Source:MGI Symbol;Acc:MGI:1891037] Location Chromosome 13: 68,560,780-68,582,149 reverse strand. GRCm38:CM001006.2 About this gene This gene has 9 transcripts (splice variants), 209 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mtrr- ENSMUST00000223398.1 3861 696aa ENSMUSP00000152387.1 Protein coding CCDS26620 A0A0R4J0G9 TSL:1 209 GENCODE basic APPRIS P1

Mtrr- ENSMUST00000045827.4 3692 696aa ENSMUSP00000039810.4 Protein coding CCDS26620 A0A0R4J0G9 TSL:1 201 GENCODE basic APPRIS P1

Mtrr- ENSMUST00000220544.1 715 190aa ENSMUSP00000152659.1 Protein coding - A0A1Y7VLV0 CDS 5' 202 incomplete TSL:3

Mtrr- ENSMUST00000220973.1 636 93aa ENSMUSP00000152561.1 Protein coding - A0A1Y7VM78 CDS 3' 203 incomplete TSL:2

Mtrr- ENSMUST00000221259.1 532 68aa ENSMUSP00000152593.1 Protein coding - A0A1Y7VJV6 CDS 3' 204 incomplete TSL:3

Mtrr- ENSMUST00000223101.1 2728 157aa ENSMUSP00000152884.1 Nonsense mediated - A0A1Y7VKC5 TSL:1 208 decay

Mtrr- ENSMUST00000222107.1 636 58aa ENSMUSP00000152837.1 Nonsense mediated - A0A1Y7VKD9 TSL:5 206 decay

Mtrr- ENSMUST00000221800.1 668 No - Retained intron - - TSL:3 205 protein

Mtrr- ENSMUST00000223055.1 664 No - Retained intron - - TSL:2 207 protein

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41.37 kb Forward strand

68.56Mb 68.57Mb 68.58Mb 68.59Mb Genes Fastkd3-206 >protein coding (Comprehensive set...

Fastkd3-204 >lncRNA

Fastkd3-201 >protein coding

Fastkd3-209 >nonsense mediated decay

Fastkd3-208 >protein coding

Fastkd3-205 >protein coding

Fastkd3-202 >retained intron

Fastkd3-207 >nonsense mediated decay

Fastkd3-203 >protein coding

Contigs CT033781.12 > Genes < Mtrr-201protein coding < Gm48556-201lncRNA (Comprehensive set...

< Mtrr-208nonsense mediated decay

< Mtrr-209protein coding

< Mtrr-202protein coding < Mtrr-206nonsense mediated decay

< Mtrr-207retained intron

< Mtrr-203protein coding

< Mtrr-204protein coding

< Mtrr-205retained intron

Regulatory Build

68.56Mb 68.57Mb 68.58Mb 68.59Mb Reverse strand 41.37 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000045827

< Mtrr-201protein coding

Reverse strand 21.34 kb

ENSMUSP00000039... Low complexity (Seg) Superfamily Flavoprotein-like superfamily Riboflavin synthase-like beta-barrel Ferredoxin-NADP reductase (FNR), nucleotide-binding domain

Prints Flavodoxin-like Flavoprotein pyridine nucleotide cytochrome reductase Pfam Flavodoxin/nitric oxide synthase Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding

Oxidoreductase FAD/NAD(P)-binding PROSITE profiles Flavodoxin/nitric oxide synthase FAD-binding domain, ferredoxin reductase-type

PANTHER PTHR19384

PTHR19384:SF84 Gene3D Flavoprotein-like superfamily 2.40.30.10 Ferredoxin-NADP reductase (FNR), nucleotide-binding domain

NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily CDD cd06203

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

Variant Legend

stop gained inframe deletion missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 696

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

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