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Mouse Sertad1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Sertad1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Sertad1 gene (NCBI Reference Sequence: NM_018820 ; Ensembl: ENSMUSG00000008384 ) is located on Mouse chromosome 7. 2 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000008528). 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 Sertad1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-104H22 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 knock-out allele exhibit impaired glucose tolerance and decreased glucose-stimulated insulin secretion associated with decreased islet number and beta cell mass.

Exon 2 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 2. The size of effective cKO region: ~1814 bp. The cKO 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Sertad1 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(6708bp) | A(22.05% 1479) | C(26.76% 1795) | T(23.69% 1589) | G(27.5% 1845)

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% chr7 + 27486257 27489256 3000 browser details YourSeq 84 2249 2767 3000 92.0% chr10 + 80660309 81061830 401522 browser details YourSeq 67 1548 2048 3000 71.5% chr2 - 119085429 119085815 387 browser details YourSeq 46 2664 2764 3000 91.1% chr1 - 151555629 151555883 255 browser details YourSeq 46 2651 2764 3000 85.8% chr1 - 135220559 135220670 112 browser details YourSeq 45 2228 2520 3000 65.4% chr5 + 100233821 100233983 163 browser details YourSeq 44 2251 2324 3000 89.8% chr14 + 55082209 55082280 72 browser details YourSeq 43 2390 2520 3000 89.4% chr7 - 31096468 31096596 129 browser details YourSeq 43 2197 2272 3000 77.4% chr1 - 133943319 133943383 65 browser details YourSeq 41 2709 2764 3000 87.5% chr9 - 66160796 66160852 57 browser details YourSeq 40 36 205 3000 91.2% chr3 - 108352685 108352853 169 browser details YourSeq 40 2232 2324 3000 93.5% chr18 + 15398926 15399023 98 browser details YourSeq 39 2736 2792 3000 93.4% chr12 + 87293010 87293358 349 browser details YourSeq 38 2708 2763 3000 88.1% chr8 - 80930630 80930683 54 browser details YourSeq 37 2228 2764 3000 57.5% chr5 - 139601054 139601437 384 browser details YourSeq 37 2233 2323 3000 91.2% chr11 - 68252687 68252801 115 browser details YourSeq 36 2232 2275 3000 92.2% chr6 - 50298387 50298429 43 browser details YourSeq 36 2229 2274 3000 90.0% chr2 - 165025695 165025739 45 browser details YourSeq 36 2229 2276 3000 90.0% chr2 + 139997040 139997086 47 browser details YourSeq 35 1983 2275 3000 97.4% chr2 + 163274488 163274858 371

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% chr7 + 27489965 27492964 3000 browser details YourSeq 138 1614 2779 3000 88.8% chr17 + 23678309 23842848 164540 browser details YourSeq 84 2682 2795 3000 86.9% chr4 + 119162518 119162631 114 browser details YourSeq 80 2722 2898 3000 92.6% chr4 + 44335668 44336081 414 browser details YourSeq 78 2686 2787 3000 88.3% chrX + 58009937 58010038 102 browser details YourSeq 78 2694 2787 3000 91.5% chr2 + 25188546 25188639 94 browser details YourSeq 75 2691 2787 3000 88.7% chr6 - 35953460 35953556 97 browser details YourSeq 72 2694 2787 3000 88.3% chr13 + 45262668 45262761 94 browser details YourSeq 71 2684 2782 3000 88.9% chr18 - 42365864 42365964 101 browser details YourSeq 71 2702 2787 3000 91.9% chr5 + 73310760 73310847 88 browser details YourSeq 70 2680 2787 3000 80.4% chr4 - 119065165 119065271 107 browser details YourSeq 70 2694 2787 3000 87.3% chr12 + 55517125 55517218 94 browser details YourSeq 70 2694 2787 3000 87.3% chr11 + 106466865 106466958 94 browser details YourSeq 68 2694 2787 3000 86.2% chr19 - 25441762 25441855 94 browser details YourSeq 67 2694 2788 3000 85.3% chr1 - 118552721 118552815 95 browser details YourSeq 67 2694 2787 3000 90.4% chr1 + 71621385 71621479 95 browser details YourSeq 66 2691 2779 3000 87.7% chr4 - 155836072 155836161 90 browser details YourSeq 66 2691 2787 3000 81.1% chr2 + 29728605 29728699 95 browser details YourSeq 66 2694 2777 3000 89.3% chr11 + 74750550 74750633 84 browser details YourSeq 65 2692 2787 3000 84.4% chr16 - 92677079 92677176 98

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: Sertad1 SERTA domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 55942, updated on 12-Aug-2019

Gene summary

Official Symbol Sertad1 provided by MGI Official Full Name SERTA domain containing 1 provided by MGI Primary source MGI:MGI:1913438 See related Ensembl:ENSMUSG00000008384 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 Sei1; Sei-1; AV000455; AW456625; TRIP-Br1; 1110032C13Rik Expression Ubiquitous expression in small intestine adult (RPKM 40.2), colon adult (RPKM 32.4) and 26 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 A3 See Sertad1 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (27486953..27490316)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (28271972..28275333)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 2 transcripts

Gene: Sertad1 ENSMUSG00000008384

Description SERTA domain containing 1 [Source:MGI Symbol;Acc:MGI:1913438] Gene Synonyms 1110032C13Rik, Sei-1, Trip-Br1, p34SEI-1 Location Chromosome 7: 27,486,910-27,490,316 forward strand. GRCm38:CM001000.2 About this gene This gene has 2 transcripts (splice variants), 82 orthologues, 1 paralogue, is a member of 2 Ensembl protein families and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Sertad1-201 ENSMUST00000008528.7 1233 236aa ENSMUSP00000008528.7 Protein coding CCDS21022 Q9JL10 TSL:1 GENCODE basic APPRIS P1

Sertad1-202 ENSMUST00000135881.1 366 84aa ENSMUSP00000119180.1 Protein coding - D3Z2X7 CDS 3' incomplete TSL:3

23.41 kb Forward strand

27.48Mb 27.49Mb 27.50Mb Genes (Comprehensive set... Sertad3-201 >protein coding Sertad1-201 >protein coding Prx-206 >protein coding

Sertad1-202 >protein coding Prx-205 >protein coding

Prx-203 >protein coding

Prx-202 >protein coding

Contigs < AC158304.11 Regulatory Build

27.48Mb 27.49Mb 27.50Mb Reverse strand 23.41 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

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Transcript: ENSMUST00000008528

3.41 kb Forward strand

Sertad1-201 >protein coding

ENSMUSP00000008... MobiDB lite Low complexity (Seg) Pfam SERTA domain PROSITE profiles SERTA domain PANTHER PTHR16277:SF12

PTHR16277

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 200 236

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

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