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Mouse Dhrs4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dhrs4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dhrs4 gene (NCBI Reference Sequence: NM_001037938 ; Ensembl: ENSMUSG00000022210 ) is located on Mouse chromosome 14. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000022821). 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 Dhrs4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-158G18 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 exhibit a decreased mean serum IgG2a response to ovalbumin challenge when compared with that of controls. Female mutants exhibit an increased depressive-like response during tail suspension testing.

Exon 2 starts from about 15.77% 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: 992 bp, and the size of intron 2 for 3'-loxP site insertion: 5913 bp. The size of effective cKO region: ~678 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 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Dhrs4 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(7178bp) | A(26.89% 1930) | C(23.42% 1681) | T(25.26% 1813) | G(24.44% 1754)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr14 + 55476777 55479776 3000 browser details YourSeq 237 538 1265 3000 83.4% chr17 - 27936497 28276033 339537 browser details YourSeq 236 584 1277 3000 83.5% chr1 + 57877805 57987340 109536 browser details YourSeq 191 989 1338 3000 82.4% chr17 + 29658071 29658432 362 browser details YourSeq 187 975 1359 3000 84.2% chr12 - 12789056 12789440 385 browser details YourSeq 169 995 1296 3000 84.8% chr19 - 26291467 26291778 312 browser details YourSeq 125 577 774 3000 89.4% chr6 + 145261272 145261472 201 browser details YourSeq 123 1015 1350 3000 84.3% chr17 - 33117614 33117951 338 browser details YourSeq 123 595 776 3000 89.3% chr4 + 140900961 140901148 188 browser details YourSeq 123 975 1275 3000 87.3% chr10 + 44270782 44271082 301 browser details YourSeq 120 1110 1357 3000 88.5% chr13 - 112018981 112019234 254 browser details YourSeq 119 576 771 3000 90.0% chr16 + 36496534 36496729 196 browser details YourSeq 117 568 733 3000 87.4% chr11 - 20709102 20709269 168 browser details YourSeq 117 595 774 3000 92.2% chr2 + 157491109 157491294 186 browser details YourSeq 116 584 718 3000 93.3% chr15 - 79009279 79009416 138 browser details YourSeq 116 577 774 3000 89.1% chr4 + 31818963 31819163 201 browser details YourSeq 115 577 773 3000 91.4% chr2 - 128646734 128646959 226 browser details YourSeq 115 584 769 3000 90.8% chr7 + 111418412 111418613 202 browser details YourSeq 112 602 774 3000 85.3% chr12 + 10658013 10658183 171 browser details YourSeq 111 577 774 3000 82.7% chr2 - 76148899 76149081 183

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% chr14 + 55480455 55483454 3000 browser details YourSeq 974 2002 3000 3000 98.8% chr1 - 15866880 15867878 999 browser details YourSeq 974 2003 3000 3000 98.5% chr3 + 7790687 7791682 996 browser details YourSeq 966 2001 3000 3000 98.0% chr12 - 23162565 23163562 998 browser details YourSeq 965 2002 3000 3000 98.2% chr5 + 3251614 3252611 998 browser details YourSeq 963 2003 3000 3000 97.8% chr4 + 4236962 4237950 989 browser details YourSeq 961 2003 3000 3000 98.3% chr9 - 19210734 19211733 1000 browser details YourSeq 961 2002 3000 3000 98.3% chr9 + 50626280 50627279 1000 browser details YourSeq 960 2002 3000 3000 97.7% chr18 + 87932971 87933961 991 browser details YourSeq 959 2002 3000 3000 97.7% chr18 + 9889169 9890165 997 browser details YourSeq 959 2003 3000 3000 97.7% chr10 + 32960212 32961204 993 browser details YourSeq 959 2002 3000 3000 98.1% chr1 + 60786322 60787321 1000 browser details YourSeq 957 2002 3000 3000 97.6% chr13 - 90290337 90291333 997 browser details YourSeq 957 2003 3000 3000 98.0% chr13 + 23324530 23325525 996 browser details YourSeq 956 1993 3000 3000 97.5% chrX - 38717461 38718467 1007 browser details YourSeq 956 2003 3000 3000 97.5% chr7 - 25574196 25575190 995 browser details YourSeq 954 2003 3000 3000 97.5% chr13 + 94080547 94081533 987 browser details YourSeq 953 2003 3000 3000 97.9% chr2 + 14345370 14346369 1000 browser details YourSeq 949 2002 3000 3000 97.1% chr4 - 10433390 10434376 987 browser details YourSeq 949 2002 3000 3000 97.9% chr2 - 120800803 120801801 999

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: Dhrs4 dehydrogenase/reductase (SDR family) member 4 [ Mus musculus (house mouse) ] Gene ID: 28200, updated on 24-Oct-2019

Gene summary

Official Symbol Dhrs4 provided by MGI Official Full Name dehydrogenase/reductase (SDR family) member 4 provided by MGI Primary source MGI:MGI:90169 See related Ensembl:ENSMUSG00000022210 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 CR; RRD; NDRD; PHCR; PSCD; mouNRDR; AI043103; AI790593; D14Ucla2 Expression Ubiquitous expression in kidney adult (RPKM 28.9), liver E18 (RPKM 24.8) and 28 other tissues See more

Genomic context

Location: 14 C3; 14 28.19 cM See Dhrs4 in Genome Data Viewer Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (55478741..55490340)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (56097595..56109177)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 8 transcripts

Gene: Dhrs4 ENSMUSG00000022210

Description dehydrogenase/reductase (SDR family) member 4 [Source:MGI Symbol;Acc:MGI:90169] Gene Synonyms D14Ucla2, RRD Location Chromosome 14: 55,478,758-55,490,340 forward strand. GRCm38:CM001007.2 About this gene This gene has 8 transcripts (splice variants), 307 orthologues, 13 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dhrs4- ENSMUST00000022821.7 1392 279aa ENSMUSP00000022821.6 Protein coding CCDS27111 Q99LB2 TSL:1 201 GENCODE basic APPRIS P1

Dhrs4- ENSMUST00000226168.1 828 216aa ENSMUSP00000154481.1 Protein coding - Q9D2U3 GENCODE basic 202

Dhrs4- ENSMUST00000228353.1 692 194aa ENSMUSP00000153847.1 Protein coding - A0A2I3BPY3 CDS 5' incomplete 208

Dhrs4- ENSMUST00000226298.1 626 76aa ENSMUSP00000154666.1 Protein coding - A0A2I3BRQ8 CDS 5' incomplete 203

Dhrs4- ENSMUST00000226902.1 537 179aa ENSMUSP00000154474.1 Protein coding - A0A2I3BRG8 CDS 5' and 3' 204 incomplete

Dhrs4- ENSMUST00000227488.1 498 124aa ENSMUSP00000154006.1 Protein coding - A0A2I3BQ25 CDS 3' incomplete 205

Dhrs4- ENSMUST00000227542.1 831 No - Retained - - - 207 protein intron

Dhrs4- ENSMUST00000227513.1 762 No - Retained - - - 206 protein intron

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31.58 kb Forward strand 55.47Mb 55.48Mb 55.49Mb 55.50Mb Genes Dhrs4-201 >protein coding Carmil3-212 >nonsense mediated decay (Comprehensive set...

Dhrs4-205 >protein coding Dhrs4-207 >retained intron Carmil3-210 >retained intron

Dhrs4-202 >protein coding Carmil3-205 >nonsense mediated decay

Dhrs4-206 >retained intron Carmil3-201 >protein coding

Dhrs4-204 >protein coding Carmil3-209 >retained intron

Dhrs4-208 >protein coding Carmil3-208 >retained intron

Dhrs4-203 >protein coding Carmil3-206 >lncRNA

Carmil3-203 >nonsense mediated decay

Carmil3-202 >retained intron

Carmil3-211 >retained intron

Contigs < AC159002.2 Regulatory Build

55.47Mb 55.48Mb 55.49Mb 55.50Mb Reverse strand 31.58 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000022821

11.58 kb Forward strand

Dhrs4-201 >protein coding

ENSMUSP00000022... Low complexity (Seg) Superfamily NAD(P)-binding domain superfamily Prints Short-chain dehydrogenase/reductase SDR

Short-chain dehydrogenase/reductase SDR Pfam PF13561 PROSITE patterns Short-chain dehydrogenase/reductase, conserved site PANTHER Dehydrogenase/reductase SDR member 4-like

PTHR43943 Gene3D 3.40.50.720 CDD cd08936

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 279

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

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