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Mouse Yju2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Yju2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Yju2 (NCBI Reference Sequence: NM_028381.3 ; Ensembl: ENSMUSG00000003208 ) is located on Mouse 17. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000086869). 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 Yju2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-347C23 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 2 starts from about 2.65% 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: 1451 bp, and the size of intron 2 for 3'-loxP site insertion: 1205 bp. The size of effective cKO region: ~601 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 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Yju2 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(7101bp) | A(25.8% 1832) | C(25.07% 1780) | T(23.31% 1655) | G(25.83% 1834)

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 + 55957464 55960463 3000 browser details YourSeq 231 543 1002 3000 92.4% chr9 + 114488677 114666882 178206 browser details YourSeq 210 536 1008 3000 91.1% chr11 + 98634685 98825644 190960 browser details YourSeq 181 493 1423 3000 80.7% chr4 - 136301029 136301314 286 browser details YourSeq 174 601 993 3000 82.7% chr8 + 84276178 84276431 254 browser details YourSeq 150 529 997 3000 91.7% chr16 + 10988861 11249983 261123 browser details YourSeq 146 536 1364 3000 83.1% chr13 + 104836613 104836886 274 browser details YourSeq 144 540 1024 3000 84.3% chr6 - 27356657 27356847 191 browser details YourSeq 144 647 992 3000 91.4% chr10 - 122996720 122997123 404 browser details YourSeq 140 662 997 3000 87.9% chr10 - 63387415 63387735 321 browser details YourSeq 137 597 1002 3000 82.3% chr4 - 137460286 137460516 231 browser details YourSeq 136 613 963 3000 88.2% chr6 - 124042641 124043198 558 browser details YourSeq 136 859 1237 3000 91.0% chr3 + 153894201 153894737 537 browser details YourSeq 134 851 1015 3000 91.5% chr14 + 18811077 18811245 169 browser details YourSeq 133 857 1023 3000 94.0% chrX - 140650410 140650621 212 browser details YourSeq 133 835 1007 3000 89.1% chr8 - 34638222 34638398 177 browser details YourSeq 133 857 1451 3000 79.8% chr2 - 179247225 179247383 159 browser details YourSeq 132 860 1024 3000 90.6% chr3 - 146675566 146675729 164 browser details YourSeq 132 755 998 3000 89.8% chr2 - 26427218 26427544 327 browser details YourSeq 131 853 1007 3000 92.3% chr9 - 37468984 37469138 155

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 + 55961065 55964064 3000 browser details YourSeq 181 2211 2794 3000 91.0% chr11 - 48503808 48504449 642 browser details YourSeq 168 2498 2803 3000 87.9% chr10 - 57625257 57625630 374 browser details YourSeq 167 2407 2815 3000 87.1% chr16 + 55496677 55497109 433 browser details YourSeq 151 2407 2791 3000 84.0% chr15 + 74969107 74969538 432 browser details YourSeq 150 228 1803 3000 89.1% chr11 - 52397967 52611139 213173 browser details YourSeq 146 2560 2809 3000 86.9% chr14 + 68979511 68979820 310 browser details YourSeq 144 2319 2797 3000 86.2% chr9 - 15900288 15901008 721 browser details YourSeq 143 2555 2814 3000 88.2% chr11 - 114970735 114971041 307 browser details YourSeq 142 2479 2798 3000 89.4% chr5 - 128849127 128849501 375 browser details YourSeq 136 2512 2768 3000 83.0% chr1 + 75727929 75728229 301 browser details YourSeq 131 953 1099 3000 94.6% chr17 - 45163899 45164045 147 browser details YourSeq 130 2502 2689 3000 86.1% chr2 + 181325185 181325374 190 browser details YourSeq 127 2401 2798 3000 89.6% chr2 - 119200671 119201105 435 browser details YourSeq 127 2571 2797 3000 90.0% chr12 - 112703470 112703752 283 browser details YourSeq 125 2490 2814 3000 87.5% chr7 - 105493664 105494050 387 browser details YourSeq 125 2455 2643 3000 86.5% chr9 + 115156686 115156874 189 browser details YourSeq 121 953 1099 3000 91.2% chrX + 38479719 38479865 147 browser details YourSeq 119 2552 2779 3000 87.9% chr12 + 31174171 31174414 244 browser details YourSeq 118 237 508 3000 89.5% chr13 + 75903421 75904040 620

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 information: Yju2 YJU2 splicing factor [ Mus musculus () ] Gene ID: 72886, updated on 26-Jun-2020

Gene summary

Official Symbol Yju2 provided by MGI Official Full Name YJU2 splicing factor provided by MGI Primary source MGI:MGI:1920136 See related Ensembl:ENSMUSG00000003208 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Ccdc94; AI413813; 2900016D05Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 6.6), CNS E14 (RPKM 5.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: 17; 17 D See Yju2 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (55959187..55967951)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (56098610..56107374)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 4 transcripts

Gene: Yju2 ENSMUSG00000003208

Description YJU2 splicing factor [Source:MGI Symbol;Acc:MGI:1920136] Gene Synonyms 2900016D05Rik, Ccdc94 Location Chromosome 17: 55,959,099-55,968,285 forward strand. GRCm38:CM001010.2 About this gene This gene has 4 transcripts (splice variants), 278 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Yju2-201 ENSMUST00000086869.5 1660 314aa ENSMUSP00000084082.4 Protein coding CCDS37660 Q9D6J3 TSL:1 GENCODE basic APPRIS P2

Yju2-204 ENSMUST00000233661.1 1601 256aa ENSMUSP00000156755.1 Protein coding - A0A3B2W7X7 GENCODE basic APPRIS ALT2

Yju2-202 ENSMUST00000233204.1 3557 No protein - Retained intron - - -

Yju2-203 ENSMUST00000233226.1 526 No protein - Retained intron - - -

29.19 kb Forward strand

55.95Mb 55.96Mb 55.97Mb (Comprehensive set... Ebi3-201 >protein coding Yju2-204 >protein coding Shd-201 >protein coding

Yju2-202 >retained intron Shd-206 >protein coding

Yju2-201 >protein coding Shd-205 >retained intron

Yju2-203 >retained intron Shd-202 >protein coding

Shd-207 >retained intron

Shd-203 >retained intron

Shd-204 >retained intron

Contigs AC154235.2 > Genes < Zfp119b-202protein coding < Gm16712-201antisense (Comprehensive set...

Regulatory Build

55.95Mb 55.96Mb 55.97Mb Reverse strand 29.19 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000086869

9.11 kb Forward strand

Yju2-201 >protein coding

ENSMUSP00000084... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Saf4/Yju2 protein

PANTHER Saf4/Yju2 protein

PTHR12111:SF1

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 314

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

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