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Mouse Raly Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Raly gene (NCBI Reference Sequence: NM_001139513 ; Ensembl: ENSMUSG00000027593 ) is located on Mouse chromosome 2. 9 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 8 (Transcript: ENSMUST00000116389). 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 Raly gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-73P2 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 are viable.

Exon 3 starts from about 27.46% 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: 2144 bp, and the size of intron 4 for 3'-loxP site insertion: 1881 bp. The size of effective cKO region: ~781 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 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Raly 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(7281bp) | A(27.06% 1970) | C(20.44% 1488) | T(30.28% 2205) | G(22.22% 1618)

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% chr2 + 154856429 154859428 3000 browser details YourSeq 133 1751 2180 3000 86.0% chr1 - 137125395 137125843 449 browser details YourSeq 129 1715 2151 3000 80.3% chr9 - 53892311 53892750 440 browser details YourSeq 123 1654 2141 3000 80.8% chr13 - 28433381 28433820 440 browser details YourSeq 121 2459 2780 3000 94.9% chr6 - 95208364 95208857 494 browser details YourSeq 118 1946 2146 3000 82.3% chr3 - 129106874 129107063 190 browser details YourSeq 118 1421 2169 3000 77.2% chr2 - 131744975 131745434 460 browser details YourSeq 118 1822 2218 3000 77.8% chr15 + 72577721 72577991 271 browser details YourSeq 112 2028 2219 3000 88.0% chr11 + 93258833 93259031 199 browser details YourSeq 109 2474 2774 3000 82.9% chr15 + 101469550 101469779 230 browser details YourSeq 109 2474 2770 3000 81.2% chr1 + 25105095 25105246 152 browser details YourSeq 108 1930 2180 3000 91.7% chr2 + 18580681 18580941 261 browser details YourSeq 106 1930 2175 3000 89.1% chrX - 36944465 36944711 247 browser details YourSeq 106 1953 2180 3000 87.8% chr11 + 59583690 59583922 233 browser details YourSeq 102 2474 2776 3000 80.8% chr17 - 3778254 3778379 126 browser details YourSeq 100 1983 2165 3000 85.4% chr1 + 9895131 9895322 192 browser details YourSeq 98 1740 2174 3000 89.6% chr13 - 78298136 78298570 435 browser details YourSeq 97 2474 2586 3000 94.5% chr16 + 11656778 11656962 185 browser details YourSeq 96 2477 2586 3000 95.4% chr16 + 92323317 92323442 126 browser details YourSeq 96 2474 2586 3000 93.7% chr16 + 92323352 92323470 119

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% chr2 + 154860210 154863209 3000 browser details YourSeq 149 2018 2562 3000 80.4% chr8 - 57431842 57432196 355 browser details YourSeq 118 2353 2562 3000 91.1% chr4 - 136036744 136037329 586 browser details YourSeq 104 1973 2132 3000 92.8% chr9 - 103464291 103464488 198 browser details YourSeq 103 2020 2561 3000 75.2% chr14 + 62676507 62676865 359 browser details YourSeq 100 2013 2389 3000 85.8% chr5 - 139859908 139860344 437 browser details YourSeq 100 2013 2286 3000 94.0% chr11 - 70538067 70538525 459 browser details YourSeq 99 2433 2562 3000 86.3% chr1 + 85740368 85740494 127 browser details YourSeq 93 1969 2109 3000 88.7% chr9 + 56725701 56725848 148 browser details YourSeq 92 1974 2122 3000 89.7% chr3 - 107687842 107687997 156 browser details YourSeq 90 1982 2122 3000 88.2% chr15 - 93586276 93586443 168 browser details YourSeq 88 1978 2108 3000 88.7% chr11 + 19672468 19672605 138 browser details YourSeq 87 1971 2100 3000 88.5% chr11 + 70067816 70067953 138 browser details YourSeq 86 1971 2108 3000 85.8% chr3 - 127692581 127692714 134 browser details YourSeq 85 2471 2562 3000 96.8% chr8 + 88372770 88373070 301 browser details YourSeq 84 1912 2104 3000 92.1% chr3 - 96473680 96473899 220 browser details YourSeq 84 1976 2098 3000 91.2% chr6 + 120890202 120890353 152 browser details YourSeq 83 2015 2108 3000 94.7% chr5 - 141930303 141930397 95 browser details YourSeq 83 1978 2107 3000 89.7% chr17 - 5538369 5538518 150 browser details YourSeq 83 2013 2122 3000 88.2% chr9 + 22813585 22813714 130

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: Raly hnRNP-associated with lethal yellow [ Mus musculus (house mouse) ] Gene ID: 19383, updated on 12-Aug-2019

Gene summary

Official Symbol Raly provided by MGI Official Full Name hnRNP-associated with lethal yellow provided by MGI Primary source MGI:MGI:97850 See related Ensembl:ENSMUSG00000027593 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 Merc; AI663842 Expression Ubiquitous expression in ovary adult (RPKM 41.7), liver E14 (RPKM 41.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2 H1; 2 76.83 cM See Raly in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (154790920..154871010)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (154616846..154692997)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 10 transcripts

Gene: Raly ENSMUSG00000027593

Description hnRNP-associated with lethal yellow [Source:MGI Symbol;Acc:MGI:97850] Gene Synonyms Merc Location Chromosome 2: 154,791,096-154,867,261 forward strand. GRCm38:CM000995.2 About this gene This gene has 10 transcripts (splice variants), 195 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Raly-201 ENSMUST00000029120.13 1802 312aa ENSMUSP00000029120.7 Protein coding CCDS50767 Q64012 TSL:5 GENCODE basic APPRIS ALT2

Raly-204 ENSMUST00000116389.8 1759 312aa ENSMUSP00000112090.2 Protein coding CCDS50767 Q64012 TSL:5 GENCODE basic APPRIS ALT2

Raly-202 ENSMUST00000058089.12 1747 296aa ENSMUSP00000058105.6 Protein coding CCDS16939 Q3U3F6 Q64012 TSL:1 GENCODE basic APPRIS P3

Raly-203 ENSMUST00000109701.9 1709 296aa ENSMUSP00000105323.3 Protein coding CCDS16939 Q3U3F6 Q64012 TSL:1 GENCODE basic APPRIS P3

Raly-207 ENSMUST00000140713.2 1100 298aa ENSMUSP00000119126.1 Protein coding - A2AU62 CDS 3' incomplete TSL:5

Raly-206 ENSMUST00000129137.7 851 211aa ENSMUSP00000114185.1 Protein coding - A2AU61 CDS 3' incomplete TSL:5

Raly-205 ENSMUST00000125872.7 546 141aa ENSMUSP00000119108.1 Protein coding - A2AU60 CDS 3' incomplete TSL:3

Raly-210 ENSMUST00000152038.7 1574 No protein - lncRNA - - TSL:1

Raly-208 ENSMUST00000145202.7 402 No protein - lncRNA - - TSL:3

Raly-209 ENSMUST00000151578.1 337 No protein - lncRNA - - TSL:3

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96.17 kb Forward strand 154.80Mb 154.82Mb 154.84Mb 154.86Mb Genes (Comprehensive set... Raly-203 >protein coding

Raly-206 >protein coding

Raly-204 >protein coding

Raly-205 >protein coding

Raly-201 >protein coding

Raly-202 >protein coding

Raly-207 >protein coding

Raly-210 >lncRNA

Raly-208 >lncRNA

a-204 >protein coding

Raly-209 >lncRNA

Contigs AL929024.9 > Genes < Eif2s2-201protein coding (Comprehensive set...

< Eif2s2-205protein coding

Regulatory Build

154.80Mb 154.82Mb 154.84Mb 154.86Mb Reverse strand 96.17 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000116389

76.15 kb Forward strand

Raly-204 >protein coding

ENSMUSP00000112... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily RNA-binding domain superfamily SMART RNA recognition motif domain Pfam RNA recognition motif domain PROSITE profiles RNA recognition motif domain PIRSF Heterogeneous nuclear ribonucleoprotein C PANTHER PTHR13968

PTHR13968:SF6 Gene3D Nucleotide-binding alpha-beta plait domain superfamily CDD Raly, RNA recognition motif

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

Variant Legend inframe insertion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 312

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

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