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Mouse Rasgrp3 Knockout Project (CRISPR/Cas9)

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Mouse Rasgrp3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rasgrp3 Knockout Project (CRISPR/Cas9) Objective: To create a Rasgrp3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rasgrp3 gene (NCBI Reference Sequence: NM_001166493 ; Ensembl: ENSMUSG00000071042 ) is located on Mouse chromosome 17. 17 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000164192). Exon 3~9 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: Homozygous mutant mice are viable and fertile with no obvious abnormalities in the kidneys or vasculature. Exon 3 starts from about 3.42% of the coding region. Exon 3~9 covers 48.87% of the coding region. The size of effective KO region: ~9243 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 6 7 8 9 17 Legends Exon of mouse Rasgrp3 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 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 9 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(28.65% 573) | C(22.1% 442) | T(30.7% 614) | G(18.55% 371) Note: The 2000 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(25.55% 511) | C(20.5% 410) | T(32.65% 653) | G(21.3% 426) Note: The 2000 bp section downstream of Exon 9 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. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr17 + 75492148 75494147 2000 browser details YourSeq 634 1237 1972 2000 94.1% chr12 + 71302746 71303477 732 browser details YourSeq 607 1221 1935 2000 94.8% chr9 - 85867950 85868659 710 browser details YourSeq 601 1237 1972 2000 94.3% chrX - 42448619 42449508 890 browser details YourSeq 580 1345 1971 2000 96.4% chr18 - 43269984 43270711 728 browser details YourSeq 485 1454 1972 2000 96.8% chrX - 10278297 10278815 519 browser details YourSeq 484 1454 1979 2000 96.2% chr1 + 148790772 148791307 536 browser details YourSeq 483 1454 1972 2000 96.6% chr17 - 30170149 30170667 519 browser details YourSeq 483 1454 1972 2000 96.6% chr16 - 5448170 5448688 519 browser details YourSeq 483 1454 1972 2000 96.6% chr10 - 18555697 18556215 519 browser details YourSeq 481 1454 1972 2000 96.4% chr3 - 70217437 70217955 519 browser details YourSeq 481 1454 1972 2000 96.4% chr14 - 101150374 101150892 519 browser details YourSeq 481 1454 1972 2000 96.4% chrX + 23596657 23597175 519 browser details YourSeq 481 1454 1972 2000 96.4% chr2 + 122931752 122932270 519 browser details YourSeq 481 1454 1972 2000 96.4% chr15 + 29268384 29268902 519 browser details YourSeq 481 1454 1972 2000 96.4% chr12 + 18047183 18047701 519 browser details YourSeq 480 1454 1972 2000 96.4% chr10 - 74537329 74537868 540 browser details YourSeq 479 1454 1972 2000 96.2% chr7 - 63240637 63241155 519 browser details YourSeq 479 1454 1972 2000 96.2% chr3 - 129817371 129817889 519 browser details YourSeq 479 1454 1972 2000 96.2% chr12 - 24193567 24194085 519 Note: The 2000 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% chr17 + 75503391 75505390 2000 browser details YourSeq 165 450 943 2000 84.7% chr10 - 53450730 53450912 183 browser details YourSeq 160 422 610 2000 92.2% chr18 - 35420550 35420735 186 browser details YourSeq 155 416 594 2000 93.9% chr3 - 9364966 9365147 182 browser details YourSeq 155 418 590 2000 95.3% chr2 - 119442908 119443084 177 browser details YourSeq 155 417 590 2000 94.9% chr18 + 39574317 39574492 176 browser details YourSeq 155 401 589 2000 90.7% chr17 + 44755783 44755957 175 browser details YourSeq 152 413 593 2000 94.3% chr1 + 3629099 3629286 188 browser details YourSeq 151 410 596 2000 91.4% chrX - 139466371 139466561 191 browser details YourSeq 150 450 680 2000 93.2% chr15 - 99123073 99123316 244 browser details YourSeq 150 401 590 2000 94.7% chr2 + 178863363 178863558 196 browser details YourSeq 148 433 623 2000 93.6% chr16 + 44732792 44732985 194 browser details YourSeq 147 418 581 2000 93.3% chr17 - 56031719 56031880 162 browser details YourSeq 147 397 590 2000 88.8% chr1 + 136702625 136702784 160 browser details YourSeq 144 448 611 2000 94.5% chr18 - 66717627 66717791 165 browser details YourSeq 144 419 585 2000 90.9% chr16 - 94727130 94727293 164 browser details YourSeq 144 429 591 2000 94.5% chr13 - 5266617 5266799 183 browser details YourSeq 143 430 590 2000 95.6% chr4 + 45505065 45505227 163 browser details YourSeq 142 436 594 2000 95.0% chr10 - 24627106 24627265 160 browser details YourSeq 140 438 593 2000 92.2% chr5 - 139437083 139437234 152 Note: The 2000 bp section downstream of Exon 9 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Rasgrp3 RAS, guanyl releasing protein 3 [ Mus musculus (house mouse) ] Gene ID: 240168, updated on 24-Oct-2019 Gene summary Official Symbol Rasgrp3 provided by MGI Official Full Name RAS, guanyl releasing protein 3 provided by MGI Primary source MGI:MGI:3028579 See related Ensembl:ENSMUSG00000071042 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 Gm327; BC066069 Expression Ubiquitous expression in lung adult (RPKM 6.2), heart adult (RPKM 3.5) and 24 other tissues See more Orthologs human all Genomic context Location: 17; 17 E2 See Rasgrp3 in Genome Data Viewer Exon count: 19 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (75435870..75529054) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (75835245..75928394) Chromosome 17 - NC_000083.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Rasgrp3 ENSMUSG00000071042 Description RAS, guanyl releasing protein 3 [Source:MGI Symbol;Acc:MGI:3028579] Gene Synonyms LOC240168 Location Chromosome 17: 75,435,896-75,529,054 forward strand. GRCm38:CM001010.2 About this gene This gene has 7 transcripts (splice variants), 192 orthologues, 39 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rasgrp3-201 ENSMUST00000095204.5 4720 691aa ENSMUSP00000092828.4 Protein coding CCDS37695 Q6NZH9 TSL:1 GENCODE basic APPRIS P1 Rasgrp3-202 ENSMUST00000164192.8 4655 691aa ENSMUSP00000129393.1 Protein coding CCDS37695 Q6NZH9 TSL:1 GENCODE basic APPRIS P1 Rasgrp3-203 ENSMUST00000234011.1 518 69aa ENSMUSP00000156978.1 Protein coding - A0A3Q4EG32 CDS 3' incomplete Rasgrp3-206 ENSMUST00000234660.1 390 56aa ENSMUSP00000157183.1 Protein coding - A0A3Q4EC09 CDS 3' incomplete Rasgrp3-205 ENSMUST00000234644.1 565 No protein - lncRNA - - - Rasgrp3-207 ENSMUST00000235103.1 439 No protein - lncRNA - - - Rasgrp3-204 ENSMUST00000234640.1 391 No protein - lncRNA - - - Page 7 of 9 https://www.alphaknockout.com 113.16 kb Forward strand 75.44Mb 75.46Mb 75.48Mb 75.50Mb 75.52Mb Genes (Comprehensive set... Rasgrp3-207 >lncRNA Rasgrp3-202 >protein coding Rasgrp3-204 >lncRNA Rasgrp3-201 >protein coding Rasgrp3-206 >protein coding Rasgrp3-205 >lncRNA Rasgrp3-203 >protein coding Contigs < AC118018.20 < CT010501.6 Genes < Fam98a-202retained intron (Comprehensive set... < Fam98a-201protein coding < Fam98a-204protein coding Regulatory Build 75.44Mb 75.46Mb 75.48Mb 75.50Mb 75.52Mb Reverse strand 113.16 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 Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000164192 93.13 kb Forward strand Rasgrp3-202 >protein coding ENSMUSP00000129..
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