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Mouse Rasgrf2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rasgrf2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rasgrf2 gene (NCBI Reference Sequence: NM_009027 ; Ensembl: ENSMUSG00000021708 ) is located on Mouse chromosome 13. 34 exons are identified, with the ATG start codon in exon 1 and the GGT stop codon in exon 34 (Transcript: ENSMUST00000099326). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rasgrf2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-270O23 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 targeted null mutation exhibit decreased Il2 and TNF-alpha production in stimulated T cells. Mice homozygous for mutations in both Rasgrf1 and Rasgrf2 exhibit no additional abnormalities than those observed in the Rasgrf1 mutant mice.

Exon 2 starts from about 8.11% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 77551 bp, and the size of intron 2 for 3'-loxP site insertion: 15224 bp. The size of effective cKO region: ~607 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 2 34 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rasgrf2 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(7107bp) | A(27.11% 1927) | C(21.33% 1516) | T(30.07% 2137) | G(21.49% 1527)

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% chr13 - 92053906 92056905 3000 browser details YourSeq 194 1198 1575 3000 85.2% chr15 - 37609088 37609467 380 browser details YourSeq 190 1198 1587 3000 87.1% chr17 + 85673891 85674296 406 browser details YourSeq 181 1251 1584 3000 83.8% chr11 + 80306740 80307071 332 browser details YourSeq 179 1198 1594 3000 85.5% chr5 - 134150272 134150751 480 browser details YourSeq 179 1199 1594 3000 87.8% chr18 + 53403690 53593664 189975 browser details YourSeq 172 1205 1576 3000 83.4% chr6 - 6061776 6062141 366 browser details YourSeq 167 1198 1584 3000 83.4% chr14 - 66897956 66898359 404 browser details YourSeq 161 1198 1591 3000 86.8% chr5 + 35309146 35309581 436 browser details YourSeq 159 1198 1572 3000 82.6% chrX - 150945410 150945774 365 browser details YourSeq 159 1225 1550 3000 84.4% chr11 - 109530613 109530941 329 browser details YourSeq 158 1242 1584 3000 85.3% chr5 + 99389273 99389617 345 browser details YourSeq 157 1198 1584 3000 88.7% chr7 + 122795983 122796372 390 browser details YourSeq 155 1177 1493 3000 86.4% chr10 + 43616379 43616749 371 browser details YourSeq 153 1302 1590 3000 84.9% chr5 + 113488803 113489092 290 browser details YourSeq 152 1198 1591 3000 88.4% chr9 - 9076165 9076558 394 browser details YourSeq 151 1338 1594 3000 86.8% chr13 + 64409812 64410075 264 browser details YourSeq 150 1205 1454 3000 80.7% chr16 - 13043209 13043460 252 browser details YourSeq 150 1198 1572 3000 88.2% chr12 + 71443846 71444212 367 browser details YourSeq 147 1198 1573 3000 86.9% chr4 - 141557163 141557759 597

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% chr13 - 92050299 92053298 3000 browser details YourSeq 159 458 835 3000 87.8% chr13 - 73967680 73968062 383 browser details YourSeq 150 442 758 3000 83.7% chr7 - 34917191 34917496 306 browser details YourSeq 134 497 762 3000 89.9% chr17 - 28663868 28845010 181143 browser details YourSeq 130 457 813 3000 71.8% chr17 + 26862493 26862846 354 browser details YourSeq 129 482 726 3000 89.1% chr10 + 95110321 95110573 253 browser details YourSeq 129 486 825 3000 91.7% chr1 + 92884356 92884700 345 browser details YourSeq 126 500 726 3000 89.9% chr13 - 60530509 60530740 232 browser details YourSeq 126 458 663 3000 88.2% chr3 + 34117257 34117450 194 browser details YourSeq 121 501 825 3000 84.7% chr4 - 138631261 138631578 318 browser details YourSeq 118 457 663 3000 91.7% chr11 - 116301949 116302161 213 browser details YourSeq 117 465 813 3000 91.0% chr1 + 90307687 90308044 358 browser details YourSeq 113 459 663 3000 90.1% chr8 - 82736300 82736504 205 browser details YourSeq 112 498 663 3000 92.5% chr11 + 32014725 32014897 173 browser details YourSeq 110 491 663 3000 86.6% chr1 - 89808511 89808683 173 browser details YourSeq 110 500 662 3000 90.6% chr11 + 117037685 117037850 166 browser details YourSeq 109 500 831 3000 91.7% chr17 - 29866828 29867164 337 browser details YourSeq 108 476 656 3000 92.3% chrX - 51230367 51230549 183 browser details YourSeq 108 548 823 3000 88.6% chr3 - 34507689 34508397 709 browser details YourSeq 107 497 663 3000 89.2% chr14 - 15693835 15694000 166

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rasgrf2 RAS protein-specific guanine nucleotide-releasing factor 2 [ Mus musculus (house mouse) ] Gene ID: 19418, updated on 12-Aug-2019

Gene summary

Official Symbol Rasgrf2 provided by MGI Official Full Name RAS protein-specific guanine nucleotide-releasing factor 2 provided by MGI Primary source MGI:MGI:109137 See related Ensembl:ENSMUSG00000021708 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 Grf2; AW048350; Ras-GRF2; 6330417G04Rik Annotation Annotation category: suggests misassembly information Annotation category: partial on reference assembly Expression Biased expression in frontal lobe adult (RPKM 11.9), cortex adult (RPKM 11.9) and 14 other tissues See more Orthologs human all

Genomic context

Location: 13 C3; 13 47.43 cM See Rasgrf2 in Genome Data Viewer

Exon count: 26

Annotation Status Assembly Chr Location release

GRCm38.p6 108 current 13 NC_000079.6 (91880407..92131828, complement) (GCF_000001635.26)

previous MGSCv37 NC_000079.5 (92792695..92901449, complement) , Build 37.2 13 assembly (GCF_000001635.18) (92020012..92127647, complement)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 6 transcripts

Gene: Rasgrf2 ENSMUSG00000021708

Description RAS protein-specific guanine nucleotide-releasing factor 2 [Source:MGI Symbol;Acc:MGI:109137] Gene Synonyms 6330417G04Rik, Grf2 Location Chromosome 13: 91,880,400-92,131,656 reverse strand. GRCm38:CM001006.2 About this gene This gene has 6 transcripts (splice variants), 256 orthologues, 39 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rasgrf2-205 ENSMUST00000151408.7 5797 588aa ENSMUSP00000116892.1 Protein coding - F7B9R2 CDS 5' incomplete TSL:1

Rasgrf2-201 ENSMUST00000099326.9 3567 1188aa ENSMUSP00000096930.3 Protein coding - D3Z6K8 TSL:5 GENCODE basic APPRIS P1

Rasgrf2-206 ENSMUST00000216219.1 2115 505aa ENSMUSP00000149731.1 Protein coding - A0A1L1SS23 TSL:5 GENCODE basic

Rasgrf2-202 ENSMUST00000142378.1 1789 252aa ENSMUSP00000115401.1 Protein coding - F6TYF8 CDS 5' incomplete TSL:1

Rasgrf2-203 ENSMUST00000146492.2 1323 387aa ENSMUSP00000116203.1 Protein coding - D3Z685 CDS 3' incomplete TSL:5

Rasgrf2-204 ENSMUST00000149630.7 502 168aa ENSMUSP00000115562.1 Protein coding - F6TCD3 CDS 5' and 3' incomplete TSL:5

271.26 kb Forward strand 91.9Mb 92.0Mb 92.1Mb Contigs AC167972.3 > < AC164622.3 AC146698.10 > Genes (Comprehensive set... < Ckmt2-201protein coding < Rasgrf2-202protein coding < Rasgrf2-206protein coding

< Rasgrf2-205protein coding < Rasgrf2-203protein coding

< Rasgrf2-201protein coding

< Rasgrf2-204protein coding

Regulatory Build

91.9Mb 92.0Mb 92.1Mb Reverse strand 271.26 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

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Transcript: ENSMUST00000099326

< Rasgrf2-201protein coding

Reverse strand 247.06 kb

ENSMUSP00000096... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 Dbl homology (DH) domain superfamily Ras guanine nucleotide exchange factor domain superfamily

SMART Pleckstrin homology domain Ras-like guanine nucleotide exchange factor, N-terminal

Dbl homology (DH) domain Ras guanine-nucleotide exchange factors catalytic domain Pfam Dbl homology (DH) domain Ras guanine-nucleotide exchange factors catalytic domain

Pleckstrin homology domain Ras-like guanine nucleotide exchange factor, N-terminal PROSITE profiles Pleckstrin homology domain Ras guanine-nucleotide exchange factors catalytic domain

IQ motif, EF-hand binding site Ras-like guanine nucleotide exchange factor, N-terminal

Dbl homology (DH) domain PROSITE patterns Ras guanine-nucleotide exchange factor, conserved site PANTHER Ras-like guanine nucleotide exchange factor

Ras-specific guanine nucleotide-releasing factor 2 Gene3D PH-like domain superfamily 1.20.870.10 Ras guanine-nucleotide exchange factor catalytic domain superfamily

Dbl homology (DH) domain superfamily CDD cd13261 Dbl homology (DH) domain Ras guanine-nucleotide exchange factors catalytic domain

Ras-like guanine nucleotide exchange factor, N-terminal

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

Variant Legend

stop lost missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1188

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

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