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Mouse Ro60 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ro60 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ro60 gene (NCBI Reference Sequence: NM_013835.2 ; Ensembl: ENSMUSG00000018199 ) is located on Mouse chromosome 1. 9 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000159879). 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 Ro60 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-104B10 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: Homozygous mutant mice develop symptoms similar to those observed in patients with lupus, including increased photosensitivity and membranoproliferative glomerulonephritis. The production of autoantibodies is detected in both homozygous and heterozygous mutant mice.

Exon 2 starts from the start codon. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 5894 bp, and the size of intron 2 for 3'-loxP site insertion: 3615 bp. The size of effective cKO region: ~1080 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 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ro60 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(7580bp) | A(28.36% 2150) | C(20.2% 1531) | T(31.99% 2425) | G(19.45% 1474)

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 - 143771256 143774255 3000 browser details YourSeq 92 276 474 3000 92.1% chr1 - 53227516 53227715 200 browser details YourSeq 86 160 628 3000 79.2% chr7 + 56379812 56380261 450 browser details YourSeq 78 635 913 3000 83.0% chr9 - 107040105 107040376 272 browser details YourSeq 73 438 909 3000 75.3% chr1 + 175702957 175703401 445 browser details YourSeq 70 1766 1858 3000 91.6% chr11 - 106907071 106907163 93 browser details YourSeq 69 1766 1858 3000 87.1% chr13 - 111962811 111962903 93 browser details YourSeq 67 1766 1858 3000 86.1% chr4 - 84702061 84702153 93 browser details YourSeq 66 319 927 3000 70.3% chrX - 158973489 158974005 517 browser details YourSeq 66 1775 1858 3000 89.3% chr6 - 148868368 148868451 84 browser details YourSeq 66 1775 1858 3000 89.3% chr8 + 64508240 64508323 84 browser details YourSeq 66 1766 1858 3000 83.8% chr5 + 142680072 142680152 81 browser details YourSeq 66 1775 1858 3000 89.3% chr10 + 75988697 75988780 84 browser details YourSeq 65 1766 1858 3000 86.6% chr18 - 47589485 47589579 95 browser details YourSeq 64 1775 1858 3000 88.1% chr5 + 144202232 144202315 84 browser details YourSeq 64 1775 1858 3000 88.1% chr13 + 96696169 96696252 84 browser details YourSeq 63 1775 1851 3000 91.0% chr11 - 83374712 83374788 77 browser details YourSeq 63 630 728 3000 82.5% chr7 + 56943582 56943680 99 browser details YourSeq 63 1775 1863 3000 85.4% chr5 + 149753317 149753405 89 browser details YourSeq 63 1775 1867 3000 83.9% chr11 + 77091192 77091284 93

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% chr1 - 143767176 143770175 3000 browser details YourSeq 154 1424 1896 3000 85.2% chr15 + 40828758 40829195 438 browser details YourSeq 145 1677 1856 3000 96.2% chr5 - 66540663 66540851 189 browser details YourSeq 144 1678 1838 3000 96.3% chr1 + 92328647 92328818 172 browser details YourSeq 143 1668 1852 3000 91.4% chr2 - 142018417 142018609 193 browser details YourSeq 142 1667 1842 3000 91.7% chr19 - 5834094 5834273 180 browser details YourSeq 141 2112 2620 3000 86.3% chr1 - 176043240 176043802 563 browser details YourSeq 141 1567 1835 3000 93.3% chr12 + 110530832 110531213 382 browser details YourSeq 140 1677 1852 3000 90.7% chr1 + 105630917 105631096 180 browser details YourSeq 139 1665 1852 3000 90.5% chr14 - 95773650 95773832 183 browser details YourSeq 139 1677 1831 3000 95.5% chrX + 159450484 159450646 163 browser details YourSeq 139 1677 1854 3000 88.9% chr12 + 110971542 110971712 171 browser details YourSeq 138 1668 1835 3000 93.1% chrX - 104030201 104030374 174 browser details YourSeq 138 1677 1838 3000 94.4% chr5 - 75969219 75969386 168 browser details YourSeq 138 1673 1856 3000 87.0% chr2 + 26328373 26328539 167 browser details YourSeq 138 1666 1835 3000 92.1% chr12 + 12231526 12231698 173 browser details YourSeq 138 1677 1856 3000 91.5% chr1 + 107533255 107533433 179 browser details YourSeq 138 1668 1835 3000 92.2% chr1 + 37526662 37527112 451 browser details YourSeq 137 1668 1833 3000 92.1% chr15 + 29510274 29510445 172 browser details YourSeq 136 1677 1856 3000 96.0% chr7 - 101935015 101935225 211

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: Ro60 Ro60, Y RNA binding protein [ Mus musculus (house mouse) ] Gene ID: 20822, updated on 26-Jun-2020

Gene summary

Official Symbol Ro60 provided by MGI Official Full Name Ro60, Y RNA binding protein provided by MGI Primary source MGI:MGI:106652 See related Ensembl:ENSMUSG00000018199 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 Ssa; Ssa2; Trove2; SS-A/Ro; AI646302; 1810007I17Rik; A530054J02Rik Expression Broad expression in CNS E11.5 (RPKM 5.5), CNS E18 (RPKM 5.4) and 20 other tissues See more Orthologs human all

Genomic context

Location: 1 F; 1 62.54 cM See Ro60 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 (143750790..143777063, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (145597920..145624181, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 2 transcripts

Gene: Ro60 ENSMUSG00000018199

Description Ro60, Y RNA binding protein [Source:MGI Symbol;Acc:MGI:106652] Gene Synonyms 1810007I17Rik, A530054J02Rik, SS-A/Ro, Ssa, Ssa2, Trove2 Location Chromosome 1: 143,750,790-143,777,068 reverse strand. GRCm38:CM000994.2 About this gene This gene has 2 transcripts (splice variants), 265 orthologues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ro60-202 ENSMUST00000159879.1 8738 538aa ENSMUSP00000125623.1 Protein coding CCDS15345 O08848 Q3TJ75 TSL:1 GENCODE basic APPRIS P1

Ro60-201 ENSMUST00000018343.9 4335 No protein - Retained intron - - TSL:5

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46.28 kb Forward strand 143.75Mb 143.76Mb 143.77Mb 143.78Mb Genes Glrx2-211 >retained intron Uchl5-206 >protein coding (Comprehensive set...

Glrx2-206 >protein coding Uchl5-204 >retained intron

Glrx2-210 >protein coding Uchl5-201 >protein coding

Glrx2-203 >processed transcript Uchl5-205 >retained intron

Glrx2-207 >retained intron Uchl5-202 >protein coding

Glrx2-208 >processed transcript Gm29170-201 >sense intronic

Glrx2-202 >protein coding Gm25663-201 >snoRNA

Glrx2-205 >protein coding Uchl5-207 >retained intron

Glrx2-201 >nonsense mediated decay

Glrx2-204 >protein coding

Glrx2-213 >retained intron

Glrx2-212 >retained intron

Glrx2-209 >processed transcript

Contigs AL592403.6 > Genes (Comprehensive set... < Ro60-202protein coding

< Ro60-201retained intron

Regulatory Build

143.75Mb 143.76Mb 143.77Mb 143.78Mb Reverse strand 46.28 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000159879

< Ro60-202protein coding

Reverse strand 26.28 kb

ENSMUSP00000125... Superfamily TROVE domain superfamily von Willebrand factor A-like domain superfamily

Pfam TROVE domain

PROSITE profiles TROVE domain

PANTHER 60kDa SS-A/Ro ribonucleoprotein

PTHR14202:SF0 Gene3D von Willebrand factor A-like domain superfamily CDD cd00198

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 538

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

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