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Mouse Rangap1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rangap1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rangap1 gene (NCBI Reference Sequence: NM_001146174.1 ; Ensembl: ENSMUSG00000022391 ) is located on Mouse chromosome 15. 16 exons are identified , with the ATG start codon in exon 2 and the TAG stop codon in exon 16 (Transcript: ENSMUST00000052374). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rangap1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-75O7 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: Homozygous mutation of this gene results in embryo arrest at the egg cylinder stage and lethality by E8.5. The appearance of the proamniotic cavity is delayed and epiblast cells surrounding the cavity are disorganized. Mesoderm and placental development is impaired.

Exon 3 starts from about 6.4% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 2459 bp, and the size of intron 4 for 3'-loxP site insertion: 1684 bp. The size of effective cKO region: ~1920 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level. It is possible to adjust the target region according to the actual situation during the experiment.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 3 4 5 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rangap1 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(8420bp) | A(22.95% 1932) | C(23.43% 1973) | T(28.1% 2366) | G(25.52% 2149)

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% chr15 - 81722277 81725276 3000 browser details YourSeq 447 462 2538 3000 93.3% chr11 - 86043809 86678409 634601 browser details YourSeq 360 464 2534 3000 96.5% chr1 + 136633318 136758638 125321 browser details YourSeq 354 439 2528 3000 93.2% chr10 - 128446886 128554583 107698 browser details YourSeq 352 462 2533 3000 94.5% chr10 - 41408590 41819048 410459 browser details YourSeq 338 462 2538 3000 94.5% chr11 + 53340852 53909928 569077 browser details YourSeq 312 440 2539 3000 92.9% chr10 + 39831285 40283202 451918 browser details YourSeq 307 455 2475 3000 93.8% chr12 - 56965881 57439173 473293 browser details YourSeq 302 437 2540 3000 93.7% chr11 - 5905700 6277451 371752 browser details YourSeq 176 438 998 3000 92.4% chr11 - 30982977 31112928 129952 browser details YourSeq 176 1 600 3000 83.5% chr11 + 20523694 20524094 401 browser details YourSeq 166 37 602 3000 85.3% chr10 - 76452006 76452206 201 browser details YourSeq 165 2392 2719 3000 95.7% chr1 - 152078556 152078901 346 browser details YourSeq 162 2370 2710 3000 88.7% chr15 - 70577984 70578318 335 browser details YourSeq 161 2239 2538 3000 93.6% chr7 - 3679123 3679526 404 browser details YourSeq 157 2105 2539 3000 91.6% chr1 + 34372135 34372582 448 browser details YourSeq 155 2366 2538 3000 95.9% chr10 + 101111761 101111934 174 browser details YourSeq 154 40 601 3000 84.5% chr11 - 75698888 75699135 248 browser details YourSeq 154 2365 2538 3000 95.3% chr11 - 12764938 12765112 175 browser details YourSeq 152 1 601 3000 83.9% chr11 - 119351446 119351729 284

Note: The 3000 bp section upstream of Exon 3 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% chr15 - 81717357 81720356 3000 browser details YourSeq 190 2456 2767 3000 85.7% chr7 - 59225045 59225546 502 browser details YourSeq 173 2493 2768 3000 88.5% chr11 - 95864974 96027979 163006 browser details YourSeq 172 2511 2766 3000 88.8% chr18 - 60829446 60829880 435 browser details YourSeq 163 2585 2781 3000 93.3% chr5 + 129870717 129871444 728 browser details YourSeq 162 1434 1626 3000 92.1% chr17 - 12820360 12820551 192 browser details YourSeq 161 2493 2768 3000 88.5% chr6 - 91458801 91459165 365 browser details YourSeq 155 2479 2767 3000 86.8% chr1 - 136610586 136610900 315 browser details YourSeq 154 2495 2765 3000 88.8% chr1 - 171363553 171363819 267 browser details YourSeq 150 2580 2798 3000 89.9% chr4 - 43621289 43621504 216 browser details YourSeq 150 2493 2752 3000 90.0% chr3 + 51489806 51490071 266 browser details YourSeq 150 2456 2767 3000 91.8% chr10 + 42705244 42705611 368 browser details YourSeq 149 2593 2767 3000 93.1% chr2 - 28557121 28557299 179 browser details YourSeq 149 2587 2767 3000 92.1% chr13 - 60362065 60362250 186 browser details YourSeq 148 2593 2767 3000 93.0% chr2 - 157196456 157196635 180 browser details YourSeq 147 2598 2791 3000 95.2% chr6 - 120027981 120028649 669 browser details YourSeq 147 2588 2767 3000 92.6% chr11 + 86660162 86753450 93289 browser details YourSeq 145 2580 2767 3000 91.6% chr12 - 110772905 110773274 370 browser details YourSeq 145 2580 2767 3000 90.6% chr11 - 67934432 67934625 194 browser details YourSeq 145 2587 2767 3000 92.5% chr4 + 59198867 59199056 190

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: Rangap1 RAN GTPase activating protein 1 [ Mus musculus (house mouse) ] Gene ID: 19387, updated on 25-Sep-2020

Gene summary

Official Symbol Rangap1 provided by MGI Official Full Name RAN GTPase activating protein 1 provided by MGI Primary source MGI:MGI:103071 See related Ensembl:ENSMUSG00000022391 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 Fug1; C79654; mKIAA1835 Expression Ubiquitous expression in testis adult (RPKM 67.3), CNS E11.5 (RPKM 30.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 15 E1; 15 38.21 cM See Rangap1 in Genome Data Viewer

Exon count: 18

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) 15 NC_000081.7 (81588449..81614120, complement)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (81704248..81730798, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (81534678..81560349, complement)

Chromosome 15 - NC_000081.7

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Transcript information: This gene has 7 transcripts

Gene: Rangap1 ENSMUSG00000022391

Description RAN GTPase activating protein 1 [Source:MGI Symbol;Acc:MGI:103071] Gene Synonyms Fug1 Location Chromosome 15: 81,704,248-81,745,530 reverse strand. GRCm38:CM001008.2 About this gene This gene has 7 transcripts (splice variants), 394 orthologues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rangap1- ENSMUST00000052374.12 3033 589aa ENSMUSP00000057771.5 Protein coding CCDS27670 P46061 TSL:1 201 GENCODE basic APPRIS P1

Rangap1- ENSMUST00000170134.8 2984 589aa ENSMUSP00000126849.1 Protein coding CCDS27670 P46061 TSL:1 202 GENCODE basic APPRIS P1

Rangap1- ENSMUST00000171115.2 2743 589aa ENSMUSP00000130046.1 Protein coding CCDS27670 P46061 TSL:1 203 GENCODE basic APPRIS P1

Rangap1- ENSMUST00000230302.1 990 257aa ENSMUSP00000155198.1 Protein coding - A0A2R8VHK9 CDS 3' 206 incomplete

Rangap1- ENSMUST00000230726.1 710 144aa ENSMUSP00000155851.1 Protein coding - A0A2R8W753 CDS 3' 207 incomplete

Rangap1- ENSMUST00000230287.1 1685 No - Retained - - - 205 protein intron

Rangap1- ENSMUST00000229705.1 451 No - Retained - - - 204 protein intron

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61.28 kb Forward strand

81.70Mb 81.71Mb 81.72Mb 81.73Mb 81.74Mb 81.75Mb Genes Zc3h7b-201 >protein coding (Comprehensive set...

Zc3h7b-202 >retained intron

Zc3h7b-205 >protein coding

Zc3h7b-203 >retained intron

Contigs AC102262.10 > < AC158970.3 Genes (Comprehensive set... < Chadl-201protein coding < Rangap1-202protein coding

< Chadl-204retained intron < Rangap1-201protein coding

< Rangap1-203protein coding

< Rangap1-204retained intro

< Rangap1-206protein coding

< Rangap1-205retained intron

Regulatory Build

81.70Mb 81.71Mb 81.72Mb 81.73Mb 81.74Mb 81.75Mb Reverse strand 61.28 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

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Transcript: ENSMUST00000052374

< Rangap1-201protein coding

Reverse strand 25.75 kb

ENSMUSP00000057... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF52047 Ran-GTPase activating protein 1, C-terminal domain superfamily

SMART SM00368 Pfam Leucine-rich repeat Ran-GTPase activating protein 1, C-terminal PANTHER PTHR24113:SF6

PTHR24113 Gene3D Leucine-rich repeat domain superfamily Ran-GTPase activating protein 1, C-terminal domain superfamily

CDD cd00116

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 589

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

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