https://www.alphaknockout.com

Mouse Cideb Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Cideb (NCBI Reference Sequence: NM_009894.3 ; Ensembl: ENSMUSG00000022219 ) is located on Mouse 14. 5 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 5 (Transcript: ENSMUST00000001497). Exon 2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele display a lean phenotype, increased energy expenditure and improved insulin sensitivity and are resistant to high-fat diet-induced obesity, hyperlipidemia, or liver steatosis.

Exon 2 starts from about 6.39% of the coding region. Exon 2 covers 22.07% of the coding region. The size of effective KO region: ~145 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 5

Legends Exon of mouse Cideb Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 265 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 2 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(265bp) | A(22.26% 59) | C(35.09% 93) | T(30.57% 81) | G(12.08% 32)

Note: The 265 bp section upstream of Exon 2 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(28.35% 567) | C(22.4% 448) | T(26.35% 527) | G(22.9% 458)

Note: The 2000 bp section downstream of Exon 2 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 265 1 265 265 100.0% chr14 - 55757987 55758251 265 browser details YourSeq 32 115 152 265 88.9% chr12 + 64296068 64296104 37 browser details YourSeq 29 86 124 265 96.9% chr10 - 125790753 125790792 40 browser details YourSeq 28 220 259 265 68.8% chr2 - 146682807 146682838 32 browser details YourSeq 28 187 234 265 68.8% chr15 + 80432100 80432132 33 browser details YourSeq 28 191 237 265 67.8% chr11 + 106512392 106512426 35 browser details YourSeq 25 210 236 265 96.3% chr12 - 110197200 110197226 27 browser details YourSeq 23 47 72 265 96.0% chr13 - 9295896 9295925 30 browser details YourSeq 22 130 152 265 100.0% chr15 + 5472211 5472234 24 browser details YourSeq 21 130 151 265 100.0% chr1 - 31282969 31282991 23 browser details YourSeq 21 123 143 265 100.0% chr2 + 49590358 49590378 21 browser details YourSeq 20 99 118 265 100.0% chr2 - 117485806 117485825 20 browser details YourSeq 20 132 151 265 100.0% chr16 - 13896848 13896867 20 browser details YourSeq 20 132 151 265 100.0% chr12 + 68684802 68684821 20 browser details YourSeq 20 30 49 265 100.0% chr10 + 123200415 123200434 20

Note: The 265 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 2000 1 2000 2000 100.0% chr14 - 55755842 55757841 2000 browser details YourSeq 200 978 1323 2000 87.7% chr4 - 123710391 123710864 474 browser details YourSeq 185 952 1313 2000 93.6% chr1 - 135520146 135520527 382 browser details YourSeq 172 1015 1323 2000 92.7% chr11 + 86482791 86483260 470 browser details YourSeq 169 1123 1325 2000 93.0% chr11 + 94217754 94217968 215 browser details YourSeq 167 1016 1324 2000 90.0% chr2 - 167011330 167011956 627 browser details YourSeq 163 1129 1332 2000 92.5% chr10 + 80217686 80217888 203 browser details YourSeq 161 1123 1328 2000 92.3% chr16 - 31435622 31435824 203 browser details YourSeq 159 1125 1324 2000 91.3% chr5 - 115267938 115268152 215 browser details YourSeq 158 1123 1308 2000 93.5% chr18 - 80205195 80205388 194 browser details YourSeq 158 1123 1324 2000 90.3% chr4 + 109223031 109223245 215 browser details YourSeq 157 1136 1320 2000 91.4% chr15 - 25275662 25275845 184 browser details YourSeq 157 1126 1324 2000 93.9% chr5 + 76903816 76904016 201 browser details YourSeq 157 1134 1321 2000 92.6% chr3 + 159924981 159925174 194 browser details YourSeq 157 1122 1312 2000 92.9% chr2 + 167023592 167023787 196 browser details YourSeq 156 1109 1308 2000 92.0% chr5 - 90101603 90101817 215 browser details YourSeq 156 1137 1326 2000 92.0% chr10 + 42516337 42516531 195 browser details YourSeq 155 1135 1324 2000 92.4% chr11 - 87413074 87413269 196 browser details YourSeq 155 1135 1324 2000 92.0% chr1 + 132173120 132173314 195 browser details YourSeq 155 1139 1318 2000 93.4% chr1 + 128272385 128272569 185

Note: The 2000 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: Cideb cell death-inducing DNA fragmentation factor, alpha subunit-like effector B [ Mus musculus (house mouse) ] Gene ID: 12684, updated on 26-Jun-2020

Gene summary

Official Symbol Cideb provided by MGI Official Full Name cell death-inducing DNA fragmentation factor, alpha subunit-like effector B provided by MGI Primary source MGI:MGI:1270844 See related Ensembl:ENSMUSG00000022219 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 CIDE-B; AI790179; 1110030C18Rik Expression Biased expression in small intestine adult (RPKM 216.9), liver adult (RPKM 205.1) and 8 other tissuesS ee more Orthologs human all

Genomic context

Location: 14; 14 C3 See Cideb in Genome Data Viewer Exon count: 5

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (55754052..55758424, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (56372889..56377261, complement)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 2 transcripts

Gene: Cideb ENSMUSG00000022219

Description cell death-inducing DNA fragmentation factor, alpha subunit-like effector B [Source:MGI Symbol;Acc:MGI:1270844] Gene Synonyms 1110030C18Rik, CIDE-B, DFFA-like B Location : 55,754,050-55,758,458 reverse strand. GRCm38:CM001007.2 About this gene This gene has 2 transcripts (splice variants), 226 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cideb-201 ENSMUST00000001497.8 1212 219aa ENSMUSP00000001497.7 Protein coding CCDS27127 O70303 TSL:1 GENCODE basic APPRIS P1

Cideb-202 ENSMUST00000136270.1 887 No protein - Retained intron - - TSL:2

24.41 kb Forward strand 55.745Mb 55.750Mb 55.755Mb 55.760Mb 55.765Mb Nop9-201 >protein coding Ltb4r2-201 >protein coding Ltb4r1-201 >protein coding (Comprehensive set...

Nop9-202 >retained intron Ltb4r2-202 >retained intron

Contigs AC098877.3 > Genes (Comprehensive set... < Dhrs1-201protein coding < Cideb-201protein coding

< Dhrs1-202retained intron < Cideb-202retained intron

Regulatory Build

55.745Mb 55.750Mb 55.755Mb 55.760Mb 55.765Mb Reverse strand 24.41 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000001497

< Cideb-201protein coding

Reverse strand 4.41 kb

ENSMUSP00000001... Low complexity (Seg) Superfamily SSF54277

SMART CIDE-N domain Pfam CIDE-N domain

PROSITE profiles CIDE-N domain

PANTHER PTHR12306:SF10

PTHR12306 Gene3D 3.10.20.10

CDD cd06537

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

Variant Legend

synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 219

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

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