https://www.alphaknockout.com
Mouse Rasgrp2 Knockout Project (CRISPR/Cas9)
Objective: To create a Rasgrp2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Rasgrp2 gene (NCBI Reference Sequence: NM_011242 ; Ensembl: ENSMUSG00000032946 ) is located on Mouse chromosome 19. 17 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 16 (Transcript: ENSMUST00000167240). Exon 3~12 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele do not undergo spontaneous hemorrhaging but exhibit impaired platelet aggregation, resistance to collagen-induced thrombosis, and increased bleeding times after tail transection.
Exon 3 starts from about 4.06% of the coding region. Exon 3~12 covers 73.41% of the coding region. The size of effective KO region: ~6483 bp. The KO 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'
1 3 4 5 6 7 8 9 10 11 12 17
Legends Exon of mouse Rasgrp2 Knockout region
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Overview of the Dot Plot (up) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 608 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 bp section downstream of Exon 12 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.
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Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(608bp) | A(19.9% 121) | C(29.11% 177) | T(23.85% 145) | G(27.14% 165)
Note: The 608 bp section upstream of Exon 3 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.
Overview of the GC Content Distribution (down) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(25.1% 502) | C(22.05% 441) | T(27.75% 555) | G(25.1% 502)
Note: The 2000 bp section downstream of Exon 12 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 608 1 608 608 100.0% chr19 + 6401865 6402472 608 browser details YourSeq 24 325 350 608 88.0% chr12 + 111320854 111320878 25 browser details YourSeq 23 513 536 608 100.0% chr13 - 58345574 58345604 31 browser details YourSeq 22 397 418 608 100.0% chr5 - 139932800 139932821 22 browser details YourSeq 22 28 50 608 100.0% chr1 - 60873526 60873551 26 browser details YourSeq 22 180 202 608 100.0% chr1 + 62386555 62386578 24 browser details YourSeq 20 233 252 608 100.0% chr2 - 151855333 151855352 20 browser details YourSeq 20 186 205 608 100.0% chr18 + 24179088 24179107 20
Note: The 608 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 2000 1 2000 2000 100.0% chr19 + 6408956 6410955 2000 browser details YourSeq 186 1032 1923 2000 84.8% chr19 + 5075875 5076412 538 browser details YourSeq 175 1281 1477 2000 95.4% chr9 + 31051478 31287813 236336 browser details YourSeq 170 1301 1923 2000 84.8% chr11 - 115502177 115502447 271 browser details YourSeq 169 1281 1713 2000 91.6% chr11 + 21262932 21263484 553 browser details YourSeq 163 1303 1825 2000 84.9% chr19 - 44703150 44703433 284 browser details YourSeq 162 1281 1820 2000 85.6% chr3 - 27298609 27298821 213 browser details YourSeq 161 1288 1461 2000 97.7% chr13 - 119701562 119702203 642 browser details YourSeq 155 1281 1460 2000 94.8% chr15 + 101274820 101275009 190 browser details YourSeq 154 1279 1465 2000 94.4% chr3 - 23065484 23065690 207 browser details YourSeq 154 1287 1776 2000 93.8% chr2 + 157637834 157638452 619 browser details YourSeq 153 1032 1468 2000 95.9% chr3 - 33572557 33573024 468 browser details YourSeq 153 1299 1711 2000 95.3% chr11 + 69454579 69455169 591 browser details YourSeq 152 1283 1447 2000 97.0% chr5 - 123474295 123474478 184 browser details YourSeq 151 1032 1445 2000 94.2% chr8 - 105407474 105407942 469 browser details YourSeq 151 1293 1460 2000 95.9% chr10 - 5784068 5784239 172 browser details YourSeq 151 1283 1456 2000 94.7% chr4 + 135878187 135878364 178 browser details YourSeq 150 1316 1716 2000 89.9% chr11 - 75488393 75488805 413 browser details YourSeq 150 1288 1478 2000 90.7% chr7 + 91385209 91385392 184 browser details YourSeq 150 1288 1456 2000 94.7% chr5 + 35012235 35012405 171
Note: The 2000 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Rasgrp2 RAS, guanyl releasing protein 2 [ Mus musculus (house mouse) ] Gene ID: 19395, updated on 24-Oct-2019
Gene summary
Official Symbol Rasgrp2 provided by MGI Official Full Name RAS, guanyl releasing protein 2 provided by MGI Primary source MGI:MGI:1333849 See related Ensembl:ENSMUSG00000032946 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 CDC25L; Caldaggef1; CalDAG-GEFI Expression Broad expression in spleen adult (RPKM 41.2), thymus adult (RPKM 27.3) and 24 other tissuesS ee more Orthologs human all
Genomic context
Location: 19; 19 A See Rasgrp2 in Genome Data Viewer Exon count: 24
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (6398957..6415216)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (6400583..6415216)
Chromosome 19 - NC_000085.6
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Transcript information: This gene has 24 transcripts
Gene: Rasgrp2 ENSMUSG00000032946
Description RAS, guanyl releasing protein 2 [Source:MGI Symbol;Acc:MGI:1333849] Gene Synonyms CalDAG-GEFI, Caldaggef1 Location Chromosome 19: 6,399,340-6,415,216 forward strand. GRCm38:CM001012.2 View alleles of this gene on alternative sequences About this gene This gene has 24 transcripts (splice variants), 1 gene allele, 176 orthologues, 39 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Rasgrp2- ENSMUST00000113476.7 2296 608aa ENSMUSP00000109104.1 Protein coding CCDS37898 Q9QUG9 TSL:5 208 GENCODE basic APPRIS P1
Rasgrp2- ENSMUST00000035716.14 2219 608aa ENSMUSP00000041135.8 Protein coding CCDS37898 Q9QUG9 TSL:2 201 GENCODE basic APPRIS P1
Rasgrp2- ENSMUST00000167240.7 2192 608aa ENSMUSP00000129873.1 Protein coding CCDS37898 Q9QUG9 TSL:5 224 GENCODE basic APPRIS P1
Rasgrp2- ENSMUST00000113472.7 2124 141aa ENSMUSP00000109100.1 Protein coding - Q9QUG9 TSL:1 206 GENCODE basic
Rasgrp2- ENSMUST00000138555.7 829 231aa ENSMUSP00000121635.1 Protein coding - D3Z2F9 CDS 3' 215 incomplete TSL:3
Rasgrp2- ENSMUST00000146831.7 641 164aa ENSMUSP00000120630.1 Protein coding - D3YXD6 CDS 3' 219 incomplete TSL:3
Rasgrp2- ENSMUST00000113475.7 573 141aa ENSMUSP00000109103.1 Protein coding - Q9QUG9 TSL:5 207 GENCODE basic
Rasgrp2- ENSMUST00000113471.2 535 141aa ENSMUSP00000109099.1 Protein coding - Q9QUG9 TSL:1 205 GENCODE basic
Rasgrp2- ENSMUST00000150713.7 453 95aa ENSMUSP00000120949.1 Protein coding - D3YZX2 CDS 3' 222 incomplete TSL:3
Rasgrp2- ENSMUST00000113469.2 439 70aa ENSMUSP00000109097.2 Protein coding - D3YZP1 TSL:5 204 GENCODE basic
Rasgrp2- ENSMUST00000113468.7 429 97aa ENSMUSP00000109096.1 Protein coding - D3YZP2 CDS 3' 203 incomplete TSL:5
Rasgrp2- ENSMUST00000113467.1 409 60aa ENSMUSP00000109095.1 Protein coding - D3YZP4 TSL:2 202 GENCODE basic
Rasgrp2- ENSMUST00000146601.7 352 58aa ENSMUSP00000117681.1 Protein coding - D3Z3N3 CDS 3' 218 incomplete TSL:5
Rasgrp2- ENSMUST00000127021.7 2157 70aa ENSMUSP00000119740.1 Nonsense mediated - D3YZP1 TSL:5 211 decay
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Rasgrp2- ENSMUST00000139522.7 648 125aa ENSMUSP00000123036.1 Nonsense mediated - D6RDD3 TSL:3 216 decay
Rasgrp2- ENSMUST00000152022.7 8815 No - Retained intron - - TSL:5 223 protein
Rasgrp2- ENSMUST00000135532.1 1517 No - Retained intron - - TSL:1 214 protein
Rasgrp2- ENSMUST00000149205.1 919 No - Retained intron - - TSL:1 221 protein
Rasgrp2- ENSMUST00000130480.1 660 No - Retained intron - - TSL:5 212 protein
Rasgrp2- ENSMUST00000148906.7 384 No - Retained intron - - TSL:3 220 protein
Rasgrp2- ENSMUST00000145611.1 459 No - lncRNA - - TSL:3 217 protein
Rasgrp2- ENSMUST00000133968.1 442 No - lncRNA - - TSL:5 213 protein
Rasgrp2- ENSMUST00000123661.7 430 No - lncRNA - - TSL:2 210 protein
Rasgrp2- ENSMUST00000122930.1 403 No - lncRNA - - TSL:3 209 protein
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35.88 kb Forward strand 6.39Mb 6.40Mb 6.41Mb 6.42Mb Genes (Comprehensive set... Pygm-201 >protein coding Rasgrp2-208 >protein coding Nrxn2-205 >protein coding
Pygm-202 >protein coding Rasgrp2-207 >protein codRinagsgrp2-220 >retained intron Nrxn2-201 >protein coding
Pygm-204 >retained intron Rasgrp2-203 >protein codRinagsgrp2-214 >retained intron Nrxn2-207 >retained intron
Pygm-203 >retained intron Rasgrp2-212 >retained introRnasgrp2-221 >retained intron Nrxn2-204 >protein coding
Rasgrp2-218 >protein codRinagsgrp2-223 >retained intron Nrxn2-218 >protein coding
Rasgrp2-222 >protein coding
Rasgrp2-216 >nonsense mediated decay
Rasgrp2-210 >lncRNA
Rasgrp2-206 >protein coding
Rasgrp2-217 >lncRNA
Rasgrp2-211 >nonsense mediated decay
Rasgrp2-219 >protein coding
Rasgrp2-201 >protein coding
Rasgrp2-215 >protein coding
Rasgrp2-224 >protein coding
Rasgrp2-202 >protein coding
Rasgrp2-209 >lncRNA
Rasgrp2-205 >protein coding
Rasgrp2-204 >protein coding
Rasgrp2-213 >lncRNA
Contigs AC167245.2 > Genes < Gm14965-201lncRNA (Comprehensive set...
< Gm14965-202retained intron
Regulatory Build
6.39Mb 6.40Mb 6.41Mb 6.42Mb Reverse strand 35.88 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
processed transcript RNA gene
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Transcript: ENSMUST00000167240
14.63 kb Forward strand
Rasgrp2-224 >protein coding
ENSMUSP00000129... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Ras guanine nucleotide exchange factor domain superfamily EF-hand domain pair
SSF57889 SMART Ras guanine-nucleotide exchange factors catalytic domain EF-hand domain
Ras-like guanine nucleotide exchange factor, N-terminal Protein kinase C-like, phorbol ester/diacylglycerol-binding domain Pfam Ras-like guanine nucleotide exchange factor, N-terminal Protein kinase C-like, phorbol ester/diacylglycerol-binding domain
Ras guanine-nucleotide exchange factors catalytic domain EF-hand domain PROSITE profiles Ras-like guanine nucleotide exchange factor, N-terminal Protein kinase C-like, phorbol ester/diacylglycerol-binding domain
Ras guanine-nucleotide exchange factors catalytic domain EF-hand domain PROSITE patterns EF-Hand 1, calcium-binding site
Protein kinase C-like, phorbol ester/diacylglycerol-binding domain PANTHER Ras-like guanine nucleotide exchange factor
PTHR23113:SF16 Gene3D 1.20.870.10 Ras guanine-nucleotide exchange factor catalytic domain superfamily 3.30.60.20
1.10.238.10 CDD Ras guanine-nucleotide exchange factors catalytic domain EF-hand domain
Ras-like guanine nucleotide exchange factor, N-terminal Protein kinase C-like, phorbol ester/diacylglycerol-binding domain
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend
inframe deletion missense variant splice region variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 480 540 608
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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