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Mouse Glmn Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Glmn gene (NCBI Reference Sequence: NM_133248 ; Ensembl: ENSMUSG00000029276 ) is located on Mouse chromosome 5. 19 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 19 (Transcript: ENSMUST00000078021). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Glmn gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-326I10 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 gene trap allele exhibit complete embryonic lethality during organogenesis associated with growth retardation, delayed neural tube closure, incomplete embryo turning, pericardial effusion, disorganized yolk sac vascular plexus and head mesenchyme hypocellularity.

Exon 6 starts from about 21.98% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 1309 bp, and the size of intron 6 for 3'-loxP site insertion: 1302 bp. The size of effective cKO region: ~738 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 5 6 7 19 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Glmn 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(7238bp) | A(30.35% 2197) | C(20.39% 1476) | T(27.37% 1981) | G(21.88% 1584)

Note: The sequence of homologous arms and cKO region 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 3000 1 3000 3000 100.0% chr5 - 107574173 107577172 3000 browser details YourSeq 1535 1 1571 3000 99.0% chr14 + 120785586 120787159 1574 browser details YourSeq 1534 1 1575 3000 98.8% chr11 + 8325097 8326671 1575 browser details YourSeq 1530 1 1570 3000 98.8% chr16 - 8590493 8592062 1570 browser details YourSeq 1529 1 1569 3000 98.6% chr13 - 30273538 30275105 1568 browser details YourSeq 1529 1 1567 3000 98.7% chr11 - 93427768 93429333 1566 browser details YourSeq 1527 1 1571 3000 98.7% chr2 - 70072550 70074123 1574 browser details YourSeq 1527 1 1571 3000 98.6% chr14 - 76570907 76572477 1571 browser details YourSeq 1525 1 1561 3000 98.9% chr9 - 89321380 89322940 1561 browser details YourSeq 1525 1 1569 3000 98.6% chr9 - 37881875 37883443 1569 browser details YourSeq 1525 1 1574 3000 98.6% chr3 - 8654003 8655580 1578 browser details YourSeq 1525 1 1574 3000 98.5% chr3 + 158573588 158575162 1575 browser details YourSeq 1523 1 1571 3000 98.5% chr5 - 30693469 30695039 1571 browser details YourSeq 1523 1 1574 3000 98.5% chr3 - 97143338 97144911 1574 browser details YourSeq 1523 1 1572 3000 98.3% chr1 - 84967936 84969505 1570 browser details YourSeq 1523 1 1574 3000 98.5% chr1 - 35042749 35044322 1574 browser details YourSeq 1523 1 1572 3000 98.5% chr1 + 79688886 79690458 1573 browser details YourSeq 1522 1 1574 3000 98.5% chr11 - 12772475 12774057 1583 browser details YourSeq 1522 1 1574 3000 98.5% chr4 + 88566757 88568333 1577 browser details YourSeq 1522 1 1570 3000 98.5% chr2 + 17474072 17475641 1570

Note: The 3000 bp section upstream of Exon 6 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% chr5 - 107570435 107573434 3000 browser details YourSeq 190 2177 2864 3000 88.3% chr12 + 104799576 104907315 107740 browser details YourSeq 163 2611 2913 3000 86.4% chr19 + 37709742 37710050 309 browser details YourSeq 158 2595 2893 3000 83.0% chr17 - 83053910 83054222 313 browser details YourSeq 155 2620 2944 3000 89.5% chr12 + 13014984 13015308 325 browser details YourSeq 146 2635 2913 3000 84.5% chr4 - 117956126 117956401 276 browser details YourSeq 139 2635 2913 3000 81.2% chr2 + 165335620 165335925 306 browser details YourSeq 139 2595 2944 3000 84.2% chr11 + 61379476 61379814 339 browser details YourSeq 135 2221 2862 3000 87.7% chr18 - 24175261 24470380 295120 browser details YourSeq 133 2663 2942 3000 89.9% chr2 - 131677275 131677594 320 browser details YourSeq 132 2663 2955 3000 87.8% chr18 - 60510187 60510809 623 browser details YourSeq 132 2620 2890 3000 88.9% chr1 + 13676625 13676920 296 browser details YourSeq 131 2663 2944 3000 88.7% chr9 + 106534205 106534508 304 browser details YourSeq 130 2595 2865 3000 82.9% chr9 - 55249906 55250305 400 browser details YourSeq 130 2608 2944 3000 84.0% chr14 - 121372798 121373136 339 browser details YourSeq 129 2698 2946 3000 92.2% chr1 + 36713757 36714027 271 browser details YourSeq 127 2642 2940 3000 88.3% chr15 + 87985046 87985343 298 browser details YourSeq 122 2672 2913 3000 90.7% chr8 + 4366911 4367186 276 browser details YourSeq 121 2621 2863 3000 93.0% chr3 - 56091952 56244146 152195 browser details YourSeq 121 2676 2939 3000 89.6% chr10 + 42918431 42918982 552

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Glmn glomulin, FKBP associated protein [ Mus musculus (house mouse) ] Gene ID: 170823, updated on 12-Aug-2019

Gene summary

Official Symbol Glmn provided by MGI Official Full Name glomulin, FKBP associated protein provided by MGI Primary source MGI:MGI:2141180 See related Ensembl:ENSMUSG00000029276 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 Fap48; Fap68; AW227515; 9330160J16Rik Expression Broad expression in CNS E11.5 (RPKM 4.4), CNS E18 (RPKM 3.9) and 20 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 E5 See Glmn in Genome Data Viewer

Exon count: 20

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (107548962..107597888, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (107977986..108026907, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 8 transcripts

Gene: Glmn ENSMUSG00000029276

Description glomulin, FKBP associated protein [Source:MGI Symbol;Acc:MGI:2141180] Gene Synonyms 9330160J16Rik, Fap48, Fap68 Location Chromosome 5: 107,548,967-107,597,888 reverse strand. GRCm38:CM000998.2 About this gene This gene has 8 transcripts (splice variants), 261 orthologues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Glmn- ENSMUST00000078021.12 2015 596aa ENSMUSP00000077168.6 Protein coding CCDS19505 Q8BZM1 TSL:1 201 GENCODE basic APPRIS P1

Glmn- ENSMUST00000082121.8 1945 596aa ENSMUSP00000080766.2 Protein coding CCDS19505 Q8BZM1 TSL:1 202 GENCODE basic APPRIS P1

Glmn- ENSMUST00000100949.9 1759 532aa ENSMUSP00000098509.3 Protein coding CCDS51587 Q3T9A5 TSL:1 203 GENCODE basic

Glmn- ENSMUST00000124140.1 593 100aa ENSMUSP00000123224.1 Protein coding - D3Z0L3 CDS 3' 204 incomplete TSL:5

Glmn- ENSMUST00000124546.7 2042 97aa ENSMUSP00000122129.1 Nonsense mediated - D6RGR3 TSL:5 205 decay

Glmn- ENSMUST00000137939.7 676 No - Retained intron - - TSL:2 206 protein

Glmn- ENSMUST00000148505.7 815 No - lncRNA - - TSL:5 208 protein

Glmn- ENSMUST00000143113.1 417 No - lncRNA - - TSL:5 207 protein

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68.92 kb Forward strand 107.54Mb 107.56Mb 107.58Mb 107.60Mb Genes Gm42669-201 >protein coding Rpap2-202 >protein coding (Comprehensive set...

Gm42669-202 >protein coding Rpap2-203 >protein coding

1700028K03Rik-201 >protein coding Rpap2-204 >protein coding

1700028K03Rik-206 >protein coding Rpap2-207 >lncRNA

1700028K03Rik-205 >protein coding Rpap2-201 >protein coding

1700028K03Rik-204 >lncRNA Rpap2-205 >protein coding

1700028K03Rik-202 >protein coding Rpap2-208 >protein coding

1700028K03Rik-203 >retained intron Rpap2-206 >protein coding

Contigs AC134411.7 > < AC117574.7 Genes (Comprehensive set... < Glmn-201protein coding

< Glmn-203protein coding

< Glmn-206retained intron < Glmn-208lncRNA

< Glmn-202protein coding

< Glmn-205nonsense mediated decay

< Glmn-207lncRNA

< Glmn-204protein coding

Regulatory Build

107.54Mb 107.56Mb 107.58Mb 107.60Mb Reverse strand 68.92 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000078021

< Glmn-201protein coding

Reverse strand 48.67 kb

ENSMUSP00000077... Low complexity (Seg) Pfam YAP-binding/ALF4/Glomulin

PANTHER Glomulin/ALF4

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 596

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

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