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Mouse Cmtm3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cmtm3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cmtm3 gene (NCBI Reference Sequence: NM_024217 ; Ensembl: ENSMUSG00000031875 ) is located on Mouse chromosome 8. 5 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 5 (Transcript: ENSMUST00000034343). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cmtm3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-57O3 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 26.81% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2862 bp, and the size of intron 4 for 3'-loxP site insertion: 1339 bp. The size of effective cKO region: ~2135 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 3 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cmtm3 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(8635bp) | A(23.89% 2063) | C(25.62% 2212) | T(23.74% 2050) | G(26.75% 2310)

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% chr8 + 104340509 104343508 3000 browser details YourSeq 164 1712 1905 3000 93.3% chr4 - 121007959 121008470 512 browser details YourSeq 163 1716 1905 3000 94.2% chr17 + 83565183 83565378 196 browser details YourSeq 162 1716 1907 3000 93.8% chrX - 8152255 8152507 253 browser details YourSeq 161 1666 1905 3000 91.8% chr10 - 115320985 115321232 248 browser details YourSeq 161 1714 1921 3000 88.8% chr12 + 110683876 110684080 205 browser details YourSeq 160 1713 1905 3000 92.2% chr2 - 131017534 131017735 202 browser details YourSeq 160 1715 1905 3000 93.2% chr12 - 51679331 51679896 566 browser details YourSeq 160 1714 1907 3000 92.6% chr11 + 70530064 70530261 198 browser details YourSeq 159 1714 1906 3000 90.6% chr15 + 98183622 98183813 192 browser details YourSeq 158 1725 1905 3000 95.5% chr2 + 121869085 121869277 193 browser details YourSeq 157 1729 1905 3000 94.4% chr8 - 48306647 48306823 177 browser details YourSeq 157 1713 1905 3000 91.2% chr11 - 94582090 94582284 195 browser details YourSeq 156 1715 1908 3000 91.2% chr10 - 61318963 61319175 213 browser details YourSeq 155 1712 1909 3000 92.0% chr14 - 26329629 26436599 106971 browser details YourSeq 155 1715 1906 3000 91.5% chr11 - 97620460 97620663 204 browser details YourSeq 155 1717 1905 3000 92.9% chr14 + 102369538 102369728 191 browser details YourSeq 154 1729 1927 3000 93.0% chr7 - 141121691 141121889 199 browser details YourSeq 154 1738 2038 3000 89.7% chr2 + 31715605 31715987 383 browser details YourSeq 153 1720 1906 3000 91.9% chr19 - 5325409 5325600 192

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% chr8 + 104345644 104348643 3000 browser details YourSeq 33 49 103 3000 80.0% chr8 + 69775895 69775949 55 browser details YourSeq 32 49 94 3000 84.8% chr8 - 5464446 5464491 46 browser details YourSeq 30 49 94 3000 82.7% chr8 - 106167066 106167111 46 browser details YourSeq 30 49 94 3000 82.7% chr7 - 112974116 112974161 46 browser details YourSeq 30 49 94 3000 82.7% chr5 - 33635891 33635936 46 browser details YourSeq 30 49 94 3000 82.7% chr2 - 166823371 166823416 46 browser details YourSeq 30 49 94 3000 82.7% chr1 - 45834766 45834811 46 browser details YourSeq 30 49 94 3000 82.7% chr3 + 144606146 144606191 46 browser details YourSeq 30 49 94 3000 82.7% chr11 + 73629226 73629271 46 browser details YourSeq 29 49 95 3000 80.9% chr12 + 85790561 85790607 47 browser details YourSeq 28 2532 2579 3000 69.5% chr4 - 66328952 66328991 40 browser details YourSeq 26 49 94 3000 77.5% chr1 - 108802052 108802096 45 browser details YourSeq 24 2548 2571 3000 100.0% chr14 - 76373560 76373583 24 browser details YourSeq 24 69 100 3000 87.5% chr10 + 99361795 99361826 32

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cmtm3 CKLF-like MARVEL transmembrane domain containing 3 [ Mus musculus (house mouse) ] Gene ID: 68119, updated on 12-Aug-2019

Gene summary

Official Symbol Cmtm3 provided by MGI Official Full Name CKLF-like MARVEL transmembrane domain containing 3 provided by MGI Primary source MGI:MGI:2447162 See related Ensembl:ENSMUSG00000031875 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 BNAS2; Cklfsf3; AI413895; 9430096L06Rik Expression Broad expression in ovary adult (RPKM 59.8), adrenal adult (RPKM 43.1) and 22 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 D3 See Cmtm3 in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (104339383..104347672)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (106864494..106871572)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 8 transcripts

Gene: Cmtm3 ENSMUSG00000031875

Description CKLF-like MARVEL transmembrane domain containing 3 [Source:MGI Symbol;Acc:MGI:2447162] Gene Synonyms 9430096L06Rik, BNAS2, Cklfsf3 Location Chromosome 8: 104,339,410-104,347,672 forward strand. GRCm38:CM001001.2 About this gene This gene has 8 transcripts (splice variants), 196 orthologues, 17 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cmtm3-201 ENSMUST00000034343.4 1616 184aa ENSMUSP00000034343.4 Protein coding CCDS22577 Q99LJ5 TSL:1 GENCODE basic APPRIS P1

Cmtm3-204 ENSMUST00000212081.1 850 184aa ENSMUSP00000148682.1 Protein coding CCDS22577 Q99LJ5 TSL:5 GENCODE basic APPRIS P1

Cmtm3-208 ENSMUST00000212948.1 523 142aa ENSMUSP00000148628.1 Protein coding - A0A1D5RM50 CDS 3' incomplete TSL:3

Cmtm3-205 ENSMUST00000212139.1 502 118aa ENSMUSP00000148338.1 Protein coding - A0A1D5RLE8 CDS 3' incomplete TSL:5

Cmtm3-202 ENSMUST00000211885.1 465 82aa ENSMUSP00000148513.1 Protein coding - A0A1D5RLU9 CDS 3' incomplete TSL:3

Cmtm3-207 ENSMUST00000212734.1 867 No protein - Retained intron - - TSL:2

Cmtm3-206 ENSMUST00000212399.1 790 No protein - Retained intron - - TSL:2

Cmtm3-203 ENSMUST00000211996.1 630 No protein - Retained intron - - TSL:3

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28.26 kb Forward strand

104.33Mb 104.34Mb 104.35Mb Genes (Comprehensive set... Cmtm2b-201 >protein coding Cmtm3-205 >protein coding Cmtm3-207 >retained intron

Cmtm2b-202 >protein coding Cmtm3-204 >protein coding

Cmtm3-202 >protein coding

Cmtm3-208 >protein coding

Cmtm3-203 >retained intron

Cmtm3-201 >protein coding

Cmtm3-206 >retained intron

Contigs < AC121952.4 Genes < Cmtm4-201protein coding (Comprehensive set...

Regulatory Build

104.33Mb 104.34Mb 104.35Mb Reverse strand 28.26 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000034343

7.08 kb Forward strand

Cmtm3-201 >protein coding

ENSMUSP00000034... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Marvel domain PROSITE profiles Marvel domain PANTHER PTHR22776

PTHR22776:SF3

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

Variant Legend synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 184

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

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