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Mouse Dmac1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dmac1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dmac1 (NCBI Reference Sequence: NM_025849.3 ; Ensembl: ENSMUSG00000028398 ) is located on Mouse 4. 2 are identified, with the ATG start codon in 1 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000030103). Exon 1~2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dmac1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-24E9 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 1~2 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of effective cKO region: ~1408 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele A T

5' G gRNA region 3'

1 2

Targeting vector A T G

Targeted allele A T G

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Dmac1 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(6544bp) | A(30.35% 1986) | C(20.17% 1320) | T(31.01% 2029) | G(18.47% 1209)

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% chr4 - 75278252 75281251 3000 browser details YourSeq 91 522 629 3000 88.0% chr16 + 23111333 23111432 100 browser details YourSeq 38 1996 2052 3000 81.9% chr11 - 50778549 50778601 53 browser details YourSeq 36 1996 2054 3000 86.1% chr13 + 36799316 36799373 58 browser details YourSeq 31 1998 2048 3000 81.7% chr12 - 28572835 28572886 52 browser details YourSeq 31 2297 2331 3000 88.3% chr1 + 137761711 137761744 34 browser details YourSeq 29 2297 2325 3000 100.0% chr4 + 116965121 116965149 29 browser details YourSeq 28 319 347 3000 100.0% chr13 - 108653021 108653071 51

Note: The 3000 bp section upstream of Exon 1 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% chr4 - 75274708 75277707 3000 browser details YourSeq 534 2343 3000 3000 90.9% chr6 - 61335519 61336188 670 browser details YourSeq 534 2343 3000 3000 90.9% chr11 + 19132784 19133453 670 browser details YourSeq 533 2343 3000 3000 91.3% chr3 - 37236232 37236887 656 browser details YourSeq 532 2343 3000 3000 92.0% chr11 - 6829169 6829846 678 browser details YourSeq 532 2310 3000 3000 89.8% chr6 + 104982762 104983437 676 browser details YourSeq 529 2343 3000 3000 90.7% chr10 - 75623274 75623942 669 browser details YourSeq 528 2343 3000 3000 92.0% chrX - 112322207 112322871 665 browser details YourSeq 527 2348 3000 3000 92.0% chr10 - 88367341 88367996 656 browser details YourSeq 526 2343 3000 3000 90.8% chr6 - 147556709 147557374 666 browser details YourSeq 525 2343 2991 3000 91.2% chr5 + 44407606 44408264 659 browser details YourSeq 521 2344 3000 3000 90.5% chr17 - 56527131 56527794 664 browser details YourSeq 520 2354 3000 3000 91.6% chr11 + 89881400 89882052 653 browser details YourSeq 518 2343 3000 3000 89.7% chrX + 99107813 99108477 665 browser details YourSeq 518 2343 3000 3000 91.9% chr14 + 11306243 11306913 671 browser details YourSeq 516 2343 3000 3000 90.0% chr13 + 76218464 76219113 650 browser details YourSeq 515 2343 3000 3000 90.4% chr6 - 148704319 148704974 656 browser details YourSeq 515 2343 3000 3000 91.4% chr18 + 10507164 10507828 665 browser details YourSeq 514 2343 2999 3000 92.2% chr6 - 145384685 145385343 659 browser details YourSeq 514 2343 3000 3000 89.6% chr13 + 18023430 18024098 669

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: Dmac1 distal membrane arm assembly complex 1 [ Mus musculus (house mouse) ] Gene ID: 66928, updated on 26-Jun-2020

Gene summary

Official Symbol Dmac1 provided by MGI Official Full Name distal membrane arm assembly complex 1 provided by MGI Primary source MGI:MGI:1914178 See related Ensembl:ENSMUSG00000028398 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 Tmem261; 1700027K24Rik; 3110001D03Rik Expression Ubiquitous expression in adult (RPKM 28.4), limb E14.5 (RPKM 27.6) and 28 other tissues See more Orthologs all

Genomic context

Location: 4; 4 C3 See Dmac1 in Genome Data Viewer

Exon count: 2

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (75277351..75278292, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (74923258..74924190, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 1 transcript

Gene: Dmac1 ENSMUSG00000028398

Description distal membrane arm assembly complex 1 [Source:MGI Symbol;Acc:MGI:1914178] Gene Synonyms 1700027K24Rik, 3110001D03Rik, Tmem261 Location : 75,277,354-75,278,305 reverse strand. GRCm38:CM000997.2 About this gene This gene has 1 transcript (splice variant), 98 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dmac1-201 ENSMUST00000030103.8 741 111aa ENSMUSP00000030103.8 Protein coding CCDS18288 Q9CQ00 TSL:1 GENCODE basic APPRIS P1

20.95 kb Forward strand 75.270Mb 75.275Mb 75.280Mb 75.285Mb Contigs AL773510.5 >

Genes (Comprehensive set... < Dmac1-201protein coding < Gm22864-201snoRNA

Regulatory Build

75.270Mb 75.275Mb 75.280Mb 75.285Mb Reverse strand 20.95 kb

Regulation Legend

CTCF Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000030103

< Dmac1-201protein coding

Reverse strand 952 bp

ENSMUSP00000030... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Domain of unknown function DUF4536 PANTHER PTHR36469

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

Variant Legend missense variant synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 100 111

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

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