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Mouse Msrb1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Msrb1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Msrb1 gene (NCBI Reference Sequence: NM_013759 ; Ensembl: ENSMUSG00000075705 ) is located on Mouse chromosome 17. 5 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 4 (Transcript: ENSMUST00000115262). 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 Msrb1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-363J10 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 gene trap allele exhibit oxidative stress.

Exon 2 starts from about 16.09% 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: 2736 bp, and the size of intron 2 for 3'-loxP site insertion: 432 bp. The size of effective cKO region: ~615 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 Msrb1 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(7081bp) | A(22.88% 1620) | C(23.78% 1684) | T(26.41% 1870) | G(26.93% 1907)

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% chr17 + 24736250 24739249 3000 browser details YourSeq 195 2271 2528 3000 91.6% chr2 - 180000092 180000678 587 browser details YourSeq 184 2327 2547 3000 95.2% chr6 + 118513007 118513317 311 browser details YourSeq 184 2304 2536 3000 93.0% chr2 + 154388456 154388728 273 browser details YourSeq 180 2304 2534 3000 91.7% chr9 + 72935137 72935799 663 browser details YourSeq 178 2354 2570 3000 93.7% chr5 - 142553999 142554218 220 browser details YourSeq 176 2320 2540 3000 92.8% chr13 - 37772285 37772507 223 browser details YourSeq 175 2304 2546 3000 94.0% chr11 - 100809612 100809913 302 browser details YourSeq 172 2355 2725 3000 94.4% chr12 + 102813428 102813891 464 browser details YourSeq 171 1878 2546 3000 84.4% chr17 + 24553627 24553908 282 browser details YourSeq 171 2355 2567 3000 89.8% chr1 + 142244973 142245177 205 browser details YourSeq 171 2354 2859 3000 94.4% chr1 + 131488723 131489303 581 browser details YourSeq 169 2355 2546 3000 95.3% chrX + 47321568 47321778 211 browser details YourSeq 167 2355 2536 3000 96.2% chr1 - 58575462 58575648 187 browser details YourSeq 167 2355 2545 3000 94.2% chr10 + 39901083 39901279 197 browser details YourSeq 165 2341 2538 3000 90.9% chr5 + 92497244 92497434 191 browser details YourSeq 165 2341 2529 3000 94.6% chr2 + 163869218 163869409 192 browser details YourSeq 164 2355 2540 3000 94.6% chr6 - 149524729 149524919 191 browser details YourSeq 164 2355 2536 3000 95.6% chr2 + 151658996 151659180 185 browser details YourSeq 163 2354 2544 3000 91.3% chr16 - 44336660 44336846 187

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% chr17 + 24739865 24742864 3000 browser details YourSeq 201 707 917 3000 98.2% chr6 - 37883039 37883277 239 browser details YourSeq 198 711 919 3000 98.1% chr15 - 79073502 79073718 217 browser details YourSeq 198 710 917 3000 98.1% chr13 - 86557604 86557822 219 browser details YourSeq 197 711 918 3000 97.6% chr11 - 75108196 75108407 212 browser details YourSeq 197 711 915 3000 98.6% chr4 + 34959062 34959291 230 browser details YourSeq 197 710 918 3000 97.6% chr17 + 53548465 53549074 610 browser details YourSeq 197 709 1184 3000 95.9% chr12 + 79718841 79719330 490 browser details YourSeq 196 710 913 3000 98.6% chr17 - 46461933 46462142 210 browser details YourSeq 196 711 915 3000 98.1% chr8 + 18482219 18482428 210 browser details YourSeq 196 709 917 3000 95.1% chr1 + 178135030 178135233 204 browser details YourSeq 195 708 918 3000 94.7% chr4 + 133983820 133984026 207 browser details YourSeq 194 709 917 3000 96.7% chr2 - 80843365 80843576 212 browser details YourSeq 194 611 913 3000 90.5% chr13 - 33251039 33251280 242 browser details YourSeq 194 711 913 3000 98.1% chr11 + 62693729 62693935 207 browser details YourSeq 193 711 920 3000 96.7% chr11 + 72298388 72298616 229 browser details YourSeq 192 710 910 3000 96.0% chr7 - 116113980 116114176 197 browser details YourSeq 192 698 921 3000 94.7% chr10 - 66886540 66886760 221 browser details YourSeq 192 711 908 3000 99.0% chr11 + 82963778 82964006 229 browser details YourSeq 191 711 913 3000 96.0% chr2 - 33008927 33009126 200

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: Msrb1 methionine sulfoxide reductase B1 [ Mus musculus (house mouse) ] Gene ID: 27361, updated on 24-Oct-2019

Gene summary

Official Symbol Msrb1 provided by MGI Official Full Name methionine sulfoxide reductase B1 provided by MGI Primary source MGI:MGI:1351642 See related Ensembl:ENSMUSG00000075705 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 SELX; SelR; Sepr; Sepx1; D17Wsu82e Summary The protein encoded by this gene belongs to the methionine-R-sulfoxide reductase B (MsrB) family. Members of this family Expression function as repair enzymes that protect proteins from oxidative stress by catalyzing the reduction of methionine-R-sulfoxides to methionines. This protein is highly expressed in liver and kidney, and is localized to the nucleus and cytosol. It is the only member of the MsrB family that is a selenoprotein, containing a selenocysteine (Sec) residue at its active site. It also has the highest methionine-R-sulfoxide reductase activity compared to other members containing cysteine in place of Sec. Sec is encoded by the UGA codon, which normally signals translation termination. The 3' UTRs of selenoprotein mRNAs contain a conserved stem-loop structure, designated the Sec insertion sequence (SECIS) element, that is necessary for the recognition of UGA as a Sec codon, rather than as a stop signal. Alternatively spliced transcript variants have been described for this gene. [provided by RefSeq, Oct 2016] Orthologs Ubiquitous expression in liver adult (RPKM 166.6), bladder adult (RPKM 66.9) and 25 other tissues See more human all

Genomic context

Location: 17 A3.3; 17 12.53 cM See Msrb1 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (24736593..24742778)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (24873587..24879723)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 2 transcripts

Gene: Msrb1 ENSMUSG00000075705

Description methionine sulfoxide reductase B1 [Source:MGI Symbol;Acc:MGI:1351642] Gene Synonyms D17Wsu82e, SelR, Sepr, Sepx1 Location Chromosome 17: 24,736,642-24,742,778 forward strand. GRCm38:CM001010.2 About this gene This gene has 2 transcripts (splice variants), 269 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Msrb1-201 ENSMUST00000101800.5 1211 116aa ENSMUSP00000099300.5 Protein coding CCDS28495 Q9JLC3 TSL:1 GENCODE basic APPRIS P1

Msrb1-202 ENSMUST00000115262.7 886 116aa ENSMUSP00000110917.1 Protein coding CCDS28495 Q9JLC3 TSL:1 GENCODE basic APPRIS P1

26.14 kb Forward strand 24.73Mb 24.74Mb 24.75Mb Genes (Comprehensive set... Rpl3l-202 >protein coding Msrb1-202 >protein coding

Rpl3l-203 >protein coding Msrb1-201 >protein coding

Rpl3l-201 >protein coding

Contigs < AC154566.2 Regulatory Build

24.73Mb 24.74Mb 24.75Mb Reverse strand 26.14 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

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Transcript: ENSMUST00000115262

6.14 kb Forward strand

Msrb1-202 >protein coding

ENSMUSP00000110... PDB-ENSP mappings Superfamily Mss4-like superfamily Pfam Peptide methionine sulphoxide reductase MrsB PROSITE profiles Peptide methionine sulphoxide reductase MrsB PANTHER PTHR46755:SF5

PTHR46755 Gene3D 2.170.150.20

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

Variant Legend missense variant synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 100 116

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

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