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Mouse Rap2a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rap2a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rap2a gene (NCBI Reference Sequence: NM_029519 ; Ensembl: ENSMUSG00000051615 ) is located on Mouse chromosome 14. 2 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000062117). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rap2a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-66A24 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 covers 57.19% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of intron 1 for 3'-loxP site insertion: 24560 bp. The size of effective cKO region: ~574 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 gRNA region T

5' G 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 Rap2a 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(6814bp) | A(24.13% 1644) | C(23.64% 1611) | T(26.84% 1829) | G(25.39% 1730)

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% chr14 + 120475728 120478727 3000 browser details YourSeq 56 2241 2983 3000 63.7% chr18 - 43477625 43477746 122 browser details YourSeq 36 2662 2785 3000 62.5% chrX + 94725012 94725066 55 browser details YourSeq 35 2938 2988 3000 92.7% chr12 - 69986843 69986905 63 browser details YourSeq 35 2658 2695 3000 97.4% chr11 - 120549663 120549701 39 browser details YourSeq 34 182 230 3000 82.7% chr8 + 81299754 81299801 48 browser details YourSeq 31 2662 2692 3000 100.0% chr14 - 24004082 24004112 31 browser details YourSeq 31 198 230 3000 97.0% chr13 - 3780465 3780497 33 browser details YourSeq 31 2662 2692 3000 100.0% chr7 + 34389665 34389695 31 browser details YourSeq 29 2662 2692 3000 96.8% chr5 - 23434412 23434442 31 browser details YourSeq 29 2662 2692 3000 96.8% chr2 + 74668466 74668496 31 browser details YourSeq 29 2662 2692 3000 96.8% chr2 + 18047618 18047648 31 browser details YourSeq 28 2667 2696 3000 96.7% chr4 - 119539692 119539721 30 browser details YourSeq 28 2662 2689 3000 100.0% chr10 - 57486420 57486447 28 browser details YourSeq 28 2665 2692 3000 100.0% chr6 + 52260391 52260418 28 browser details YourSeq 26 2665 2692 3000 96.5% chr14 + 55491211 55491238 28 browser details YourSeq 25 2662 2688 3000 96.3% chr7 + 112023492 112023518 27 browser details YourSeq 25 2967 2991 3000 100.0% chr18 + 77065262 77065286 25 browser details YourSeq 24 2665 2690 3000 96.2% chr2 - 152951556 152951581 26 browser details YourSeq 23 2659 2681 3000 100.0% chr8 - 108703895 108703917 23

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% chr14 + 120479292 120482291 3000 browser details YourSeq 98 1531 1746 3000 89.7% chr14 + 13994597 13994821 225 browser details YourSeq 95 1207 1659 3000 75.5% chr18 - 63672352 63672496 145 browser details YourSeq 93 1501 1633 3000 91.4% chr1 + 67081067 67081217 151 browser details YourSeq 89 1536 1678 3000 89.1% chr9 - 67949811 67949952 142 browser details YourSeq 80 1531 1678 3000 80.6% chr11 + 71731299 71731431 133 browser details YourSeq 77 1557 1678 3000 84.3% chr5 + 145239149 145239276 128 browser details YourSeq 73 1564 1659 3000 88.6% chr4 + 44629266 44629363 98 browser details YourSeq 73 1540 1630 3000 94.1% chr13 + 8844426 8844519 94 browser details YourSeq 70 1536 1659 3000 80.5% chr10 + 78026056 78026161 106 browser details YourSeq 69 1539 1659 3000 79.8% chr13 + 31301453 31301563 111 browser details YourSeq 69 1533 1659 3000 90.0% chr1 + 101692515 101692648 134 browser details YourSeq 67 1563 1693 3000 80.8% chr3 - 116278414 116278554 141 browser details YourSeq 66 1555 1659 3000 87.8% chr12 + 93272589 93272698 110 browser details YourSeq 65 1551 1678 3000 87.4% chr10 - 119053816 119053989 174 browser details YourSeq 65 1562 1659 3000 88.4% chr13 + 106831461 106831558 98 browser details YourSeq 64 1531 1680 3000 70.7% chr2 - 24963710 24963846 137 browser details YourSeq 64 1566 1678 3000 88.3% chr13 - 41254551 41254665 115 browser details YourSeq 61 1565 1658 3000 91.8% chr7 - 53375440 53375533 94 browser details YourSeq 60 1532 1630 3000 89.7% chr14 + 65253876 65253981 106

Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rap2a RAS related protein 2a [ Mus musculus (house mouse) ] Gene ID: 76108, updated on 12-Aug-2019

Gene summary

Official Symbol Rap2a provided by MGI Official Full Name RAS related protein 2a provided by MGI Primary source MGI:MGI:97855 See related Ensembl:ENSMUSG00000051615 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as 5830461H18Rik Expression Ubiquitous expression in lung adult (RPKM 22.3), frontal lobe adult (RPKM 18.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 14 E4; 14 64.72 cM See Rap2a in Genome Data Viewer

Exon count: 2

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (120478461..120507194)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (120877683..120906416)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 1 transcript

Gene: Rap2a ENSMUSG00000051615

Description RAS related protein 2a [Source:MGI Symbol;Acc:MGI:97855] Gene Synonyms 5830461H18Rik Location Chromosome 14: 120,478,444-120,507,194 forward strand. GRCm38:CM001007.2 About this gene This gene has 1 transcript (splice variant), 227 orthologues, 35 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rap2a-201 ENSMUST00000062117.13 4191 183aa ENSMUSP00000056433.6 Protein coding CCDS27341 Q80ZJ1 TSL:1 GENCODE basic APPRIS P1

48.75 kb Forward strand 120.47Mb 120.48Mb 120.49Mb 120.50Mb 120.51Mb Genes (Comprehensive set... Rap2a-201 >protein coding

Contigs CT009559.20 > AC164304.2 > Regulatory Build

120.47Mb 120.48Mb 120.49Mb 120.50Mb 120.51Mb Reverse strand 48.75 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

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Transcript: ENSMUST00000062117

28.75 kb Forward strand

Rap2a-201 >protein coding

ENSMUSP00000056... Low complexity (Seg) TIGRFAM Small GTP-binding protein domain Superfamily P-loop containing nucleoside triphosphate hydrolase SMART SM00175

SM00176

SM00173

SM00174 Prints PR00449 Pfam Small GTPase PROSITE profiles Small GTPase superfamily, Ras-type PANTHER PTHR24070:SF200

Small GTPase superfamily, Ras-type Gene3D 3.40.50.300 CDD Ras-related protein Rap2

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

Variant Legend synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 183

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

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