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Mouse Gdi1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gdi1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gdi1 gene (NCBI Reference Sequence: NM_010273 ; Ensembl: ENSMUSG00000015291 ) is located on Mouse chromosome X. 11 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000015435). Exon 2~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Gdi1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-142E5 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: Males hemizygous for a reporter allele show lower male aggression, short-term memory defects, altered synaptic vesicle pools and short-term synaptic plasticity, and impaired glutamate release. Homozygotes for a null allele show enhanced paired-pulse facilitation and sensitivity to induced seizures.

Exon 2 starts from about 3.43% of the coding region. The knockout of Exon 2~6 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1137 bp, and the size of intron 6 for 3'-loxP site insertion: 814 bp. The size of effective cKO region: ~2183 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'

10 1 2 3 4 5 6 7 8 9 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Atp6ap1 Homology arm Exon of mouse Gdi1 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(8683bp) | A(23.55% 2045) | C(23.64% 2053) | T(27.73% 2408) | G(25.07% 2177)

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% chrX + 74303330 74306329 3000 browser details YourSeq 114 29 168 3000 89.3% chr14 + 50553670 50553808 139 browser details YourSeq 40 1947 1992 3000 95.6% chr17 + 72621292 72621391 100 browser details YourSeq 35 2636 2906 3000 52.5% chr4 + 57758617 57758662 46 browser details YourSeq 35 1947 1987 3000 92.7% chr2 + 133233986 133234026 41 browser details YourSeq 34 2640 2707 3000 92.4% chr2 + 150176631 150177004 374 browser details YourSeq 31 2638 2672 3000 91.0% chr4 + 149116591 149116624 34 browser details YourSeq 29 2635 2669 3000 90.4% chr6 - 127632200 127632233 34 browser details YourSeq 28 2160 2194 3000 96.7% chr17 + 13560178 13560212 35 browser details YourSeq 27 2639 2671 3000 96.6% chr4 + 27485740 27485773 34 browser details YourSeq 26 2155 2187 3000 93.4% chr1 - 16478370 16478402 33 browser details YourSeq 25 2793 2820 3000 96.5% chr14 - 28534786 28534815 30 browser details YourSeq 23 2646 2671 3000 96.0% chr12 + 106743931 106743956 26

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% chrX + 74308513 74311512 3000 browser details YourSeq 778 1907 3000 3000 94.5% chr2 - 165665974 165666818 845 browser details YourSeq 193 1873 2540 3000 74.5% chr1 - 161297694 161298134 441 browser details YourSeq 161 1889 2416 3000 82.4% chr13 + 3564551 3565137 587 browser details YourSeq 93 2677 2789 3000 96.1% chrX - 141354627 141354794 168 browser details YourSeq 90 2674 2789 3000 96.0% chr9 + 110503046 110503174 129 browser details YourSeq 89 2677 2785 3000 89.8% chr12 + 93956790 93956894 105 browser details YourSeq 88 49 2780 3000 91.6% chr4 - 149559702 149604701 45000 browser details YourSeq 80 2675 2783 3000 96.7% chr9 - 115062319 115062457 139 browser details YourSeq 80 2680 2786 3000 90.7% chr12 + 71982298 71982400 103 browser details YourSeq 78 2689 2786 3000 94.4% chr5 - 85426349 85426446 98 browser details YourSeq 77 2673 2786 3000 91.1% chr11 + 89227240 89227351 112 browser details YourSeq 76 2677 2782 3000 93.4% chr13 - 24411251 24411386 136 browser details YourSeq 76 2680 2789 3000 81.8% chr7 + 101316355 101316436 82 browser details YourSeq 75 2688 2793 3000 87.0% chr4 + 33483759 33483857 99 browser details YourSeq 74 2678 2774 3000 93.1% chr9 + 51766695 51766809 115 browser details YourSeq 73 2675 2786 3000 90.7% chr3 + 152083263 152083384 122 browser details YourSeq 73 2683 2774 3000 91.3% chr15 + 53306046 53306135 90 browser details YourSeq 72 2677 2763 3000 85.4% chr18 + 67551355 67551429 75 browser details YourSeq 71 2680 2786 3000 87.9% chr6 - 83494751 83494852 102

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: Gdi1 guanosine diphosphate (GDP) dissociation inhibitor 1 [ Mus musculus (house mouse) ] Gene ID: 14567, updated on 12-Aug-2019

Gene summary

Official Symbol Gdi1 provided by MGI Official Full Name guanosine diphosphate (GDP) dissociation inhibitor 1 provided by MGI Primary source MGI:MGI:99846 See related Ensembl:ENSMUSG00000015291 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 GDIA; GDIalpha Expression Broad expression in CNS E14 (RPKM 251.0), whole brain E14.5 (RPKM 248.0) and 27 other tissues See more Orthologs human all

Genomic context

Location: X A7.3; X 37.97 cM See Gdi1 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (74305012..74311867)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (71550351..71557206)

Chromosome X - NC_000086.7

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Transcript information: This gene has 10 transcripts

Gene: Gdi1 ENSMUSG00000015291

Description guanosine diphosphate (GDP) dissociation inhibitor 1 [Source:MGI Symbol;Acc:MGI:99846] Gene Synonyms GDIA, Rab GDIalpha Location Chromosome X: 74,304,998-74,311,862 forward strand. GRCm38:CM001013.2 About this gene This gene has 10 transcripts (splice variants), 176 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gdi1-201 ENSMUST00000015435.10 2666 447aa ENSMUSP00000015435.4 Protein coding CCDS30227 P50396 TSL:1 GENCODE basic APPRIS P1

Gdi1-203 ENSMUST00000130581.1 667 149aa ENSMUSP00000122146.1 Protein coding - B7FAU8 CDS 5' incomplete TSL:5

Gdi1-210 ENSMUST00000153141.1 527 78aa ENSMUSP00000119805.2 Protein coding - D6RI86 CDS 3' incomplete TSL:5

Gdi1-208 ENSMUST00000146013.7 3696 No protein - Retained intron - - TSL:1

Gdi1-209 ENSMUST00000149391.7 2746 No protein - Retained intron - - TSL:2

Gdi1-206 ENSMUST00000144317.1 907 No protein - Retained intron - - TSL:3

Gdi1-205 ENSMUST00000137447.7 633 No protein - lncRNA - - TSL:2

Gdi1-202 ENSMUST00000128915.1 566 No protein - lncRNA - - TSL:5

Gdi1-207 ENSMUST00000145580.7 462 No protein - lncRNA - - TSL:1

Gdi1-204 ENSMUST00000135041.1 456 No protein - lncRNA - - TSL:1

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26.86 kb Forward strand

74.30Mb 74.31Mb 74.32Mb Genes Atp6ap1-201 >protein coding Gdi1-201 >protein coding Fam50a-201 >protein coding (Comprehensive set...

Atp6ap1-209 >protein coding Atp6ap1-207 >retained intron Gdi1-206 >retained intron Fam50a-202 >retained intron

Atp6ap1-208 >protein coding Atp6ap1-210 >protein coding Gdi1-203 >protein coding Mir7092-201 >miRNA

Atp6ap1-202 >protein coding Gdi1-208 >retained intron Fam50a-203 >retained intron

Atp6ap1-204 >retained intron Atp6ap1-206 >lncRNAGdi1-207 >lncRNA Gdi1-205 >lncRNA

Atp6ap1-203 >nonsense mediated decay Gdi1-209 >retained intron

Atp6ap1-205 >nonsense mediated decay Gdi1-210 >protein coding Gdi1-204 >lncRNA

Gdi1-202 >lncRNA

Contigs AL807376.4 > Regulatory Build

74.30Mb 74.31Mb 74.32Mb Reverse strand 26.86 kb

Regulation Legend CTCF 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: ENSMUST00000015435

6.87 kb Forward strand

Gdi1-201 >protein coding

ENSMUSP00000015... Low complexity (Seg) Superfamily FAD/NAD(P)-binding domain superfamily Prints Rab GDI protein

GDP dissociation inhibitor Pfam GDP dissociation inhibitor PANTHER PTHR11787:SF3

GDP dissociation inhibitor Gene3D 3.30.519.10

FAD/NAD(P)-binding domain superfamily

1.10.405.10

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 447

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

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