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Mouse Mtarc2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mtarc2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mtarc2 gene (NCBI Reference Sequence: NM_133684.3 ; Ensembl: ENSMUSG00000073481 ) is located on Mouse chromosome 1. 8 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 7 (Transcript: ENSMUST00000068725). 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 Mtarc2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-221J17 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 44.08% 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: 7225 bp, and the size of intron 3 for 3'-loxP site insertion: 1143 bp. The size of effective cKO region: ~669 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 3 4 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mtarc2 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. 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

Sequence 12

Summary: Full Length(7169bp) | A(27.81% 1994) | C(21.34% 1530) | T(25.39% 1820) | G(25.46% 1825)

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% chr1 - 184834279 184837278 3000 browser details YourSeq 140 1204 1385 3000 96.8% chr11 - 48791899 48792081 183 browser details YourSeq 135 2421 2591 3000 97.9% chr16 - 5442155 5442347 193 browser details YourSeq 135 1220 1372 3000 96.6% chr15 - 81935816 81935977 162 browser details YourSeq 129 1224 1364 3000 96.5% chr3 + 153699396 153699545 150 browser details YourSeq 129 1221 1371 3000 95.2% chr10 + 91113455 91113610 156 browser details YourSeq 128 95 345 3000 90.7% chr9 + 115389569 115389817 249 browser details YourSeq 127 1229 1377 3000 93.8% chr6 + 120680808 120680960 153 browser details YourSeq 126 1224 1375 3000 93.9% chr16 - 83031185 83031356 172 browser details YourSeq 126 1229 1377 3000 93.9% chr5 + 49080269 49080435 167 browser details YourSeq 126 1216 1352 3000 97.1% chr2 + 53466170 53466317 148 browser details YourSeq 126 1220 1377 3000 94.5% chr13 + 6228772 6228933 162 browser details YourSeq 120 1220 1364 3000 93.6% chr5 + 60036065 60036356 292 browser details YourSeq 118 1218 1360 3000 92.8% chr4 - 86784631 86785016 386 browser details YourSeq 118 1222 1378 3000 92.8% chr11 - 107518351 107518516 166 browser details YourSeq 118 1220 1364 3000 94.8% chr6 + 30371067 30371215 149 browser details YourSeq 115 1220 1364 3000 90.8% chr19 - 4224467 4224676 210 browser details YourSeq 115 1229 1351 3000 97.6% chr11 + 79986694 79986826 133 browser details YourSeq 113 99 493 3000 79.6% chr19 + 22566050 22566263 214 browser details YourSeq 112 1228 1368 3000 91.9% chr1 - 105524495 105524643 149

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% chr1 - 184830610 184833609 3000 browser details YourSeq 137 1856 2052 3000 84.8% chr14 + 16323963 16324158 196 browser details YourSeq 132 1863 2048 3000 84.4% chr4 + 131754799 131754983 185 browser details YourSeq 127 1880 2050 3000 85.1% chr2 + 24089876 24090043 168 browser details YourSeq 126 1863 2048 3000 82.1% chr18 - 38985221 38985404 184 browser details YourSeq 124 1852 2049 3000 87.9% chr2 - 116942820 116943048 229 browser details YourSeq 123 1866 2040 3000 83.1% chr5 - 120966632 120966803 172 browser details YourSeq 123 1856 2050 3000 85.0% chr5 - 74848351 74848544 194 browser details YourSeq 123 1858 2048 3000 86.8% chr12 - 102955290 102955478 189 browser details YourSeq 119 1883 2046 3000 86.6% chr2 - 153695045 153818356 123312 browser details YourSeq 119 1858 2050 3000 82.3% chr15 + 27102768 27102962 195 browser details YourSeq 118 1856 2050 3000 88.8% chrX - 159081004 159081198 195 browser details YourSeq 117 1866 2046 3000 82.9% chr3 - 87524187 87524363 177 browser details YourSeq 117 1866 2047 3000 81.6% chr7 + 3469826 3470006 181 browser details YourSeq 117 1877 2037 3000 87.2% chr4 + 86372733 86372895 163 browser details YourSeq 116 1863 2040 3000 85.2% chr6 + 114658766 114658941 176 browser details YourSeq 114 1854 2046 3000 87.1% chr9 - 68574125 68574350 226 browser details YourSeq 113 1856 2037 3000 87.1% chr17 - 85081466 85081649 184 browser details YourSeq 113 1863 2046 3000 88.7% chr14 + 20470902 20471084 183 browser details YourSeq 112 1897 2050 3000 84.3% chr8 - 118808672 118808823 152

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

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Gene and protein information: Mtarc2 mitochondrial amidoxime reducing component 2 [ Mus musculus (house mouse) ] Gene ID: 67247, updated on 26-Jun-2020

Gene summary

Official Symbol Mtarc2 provided by MGI Official Full Name mitochondrial amidoxime reducing component 2 provided by MGI Primary source MGI:MGI:1914497 See related Ensembl:ENSMUSG00000073481 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 Marc2; Mosc2; mARC1; 2810484M10Rik Expression Broad expression in duodenum adult (RPKM 313.8), small intestine adult (RPKM 139.6) and 19 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 H5 See Mtarc2 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (184813066..184846451, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (186636947..186669726, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 10 transcripts

Gene: Mtarc2 ENSMUSG00000073481

Description mitochondrial amidoxime reducing component 2 [Source:MGI Symbol;Acc:MGI:1914497] Gene Synonyms 2810484M10Rik, Marc2, Mosc2 Location Chromosome 1: 184,813,068-184,846,451 reverse strand. GRCm38:CM000994.2 About this gene This gene has 10 transcripts (splice variants), 229 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mtarc2-201 ENSMUST00000068725.9 1917 338aa ENSMUSP00000066715.3 Protein coding CCDS15594 Q922Q1 TSL:1 GENCODE basic APPRIS P1

Mtarc2-207 ENSMUST00000161821.3 687 190aa ENSMUSP00000125374.1 Protein coding - E0CZH6 CDS 3' incomplete TSL:3

Mtarc2-202 ENSMUST00000159293.7 931 No protein - Processed transcript - - TSL:3

Mtarc2-209 ENSMUST00000162501.6 651 No protein - Processed transcript - - TSL:5

Mtarc2-208 ENSMUST00000162261.7 633 No protein - Processed transcript - - TSL:2

Mtarc2-206 ENSMUST00000161359.7 572 No protein - Processed transcript - - TSL:3

Mtarc2-210 ENSMUST00000162866.6 475 No protein - Processed transcript - - TSL:3

Mtarc2-204 ENSMUST00000160982.1 4248 No protein - Retained intron - - TSL:1

Mtarc2-205 ENSMUST00000161341.7 968 No protein - Retained intron - - TSL:2

Mtarc2-203 ENSMUST00000160318.1 433 No protein - Retained intron - - TSL:2

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53.38 kb Forward strand 184.81Mb 184.82Mb 184.83Mb 184.84Mb 184.85Mb Contigs < AC131992.8 Genes (Comprehensive set... < Mtarc1-201protein coding < Mtarc2-201protein coding

< Mtarc1-202protein coding < Mtarc2-206processed transcript < Mtarc2-209processed transcript

< Mtarc1-203protein coding < Mtarc2-205retained intron < Mtarc2-204retained intron

< Mtarc2-208processed transcript

< Mtarc2-203retained intron < Mtarc2-207protein coding

< Mtarc2-210processed transcript

< Mtarc2-202processed transcript

< Mir1981-201miRNA

Regulatory Build

184.81Mb 184.82Mb 184.83Mb 184.84Mb 184.85Mb Reverse strand 53.38 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000068725

< Mtarc2-201protein coding

Reverse strand 32.81 kb

ENSMUSP00000066... Transmembrane heli... Low complexity (Seg) Superfamily SSF141673

Pyruvate kinase-like, insert domain superfamily Pfam MOSC, N-terminal beta barrel Molybdenum cofactor sulfurase, C-terminal

PROSITE profiles Molybdenum cofactor sulfurase, C-terminal PANTHER PTHR14237:SF27

PTHR14237

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 338

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

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