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Mouse Rap1gap2 Knockout Project (CRISPR/Cas9)

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Mouse Rap1gap2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rap1gap2 Knockout Project (CRISPR/Cas9) Objective: To create a Rap1gap2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rap1gap2 gene (NCBI Reference Sequence: NM_001015046 ; Ensembl: ENSMUSG00000038807 ) is located on Mouse chromosome 11. 25 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 24 (Transcript: ENSMUST00000102521). Exon 4~8 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 an ENU-induced allele exhibit normal blood lymphocyte populations. Exon 4 starts from about 5.1% of the coding region. Exon 4~8 covers 20.18% of the coding region. The size of effective KO region: ~6085 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 4 5 6 7 8 25 Legends Exon of mouse Rap1gap2 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 4 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 8 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(21.25% 425) | C(21.6% 432) | T(32.5% 650) | G(24.65% 493) Note: The 2000 bp section upstream of Exon 4 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(22.8% 456) | C(22.7% 454) | T(29.65% 593) | G(24.85% 497) Note: The 2000 bp section downstream of Exon 8 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% chr11 - 74441786 74443785 2000 browser details YourSeq 39 117 157 2000 97.6% chr3 - 10402613 10402653 41 browser details YourSeq 38 125 187 2000 77.3% chr5 + 142125352 142125403 52 browser details YourSeq 38 115 167 2000 93.2% chr14 + 59584314 59584367 54 browser details YourSeq 37 115 167 2000 88.4% chr9 - 34954981 34955032 52 browser details YourSeq 36 125 164 2000 95.0% chr14 - 24132777 24132816 40 browser details YourSeq 36 129 169 2000 95.0% chr5 + 129156570 129156611 42 browser details YourSeq 34 128 171 2000 97.3% chr1 + 126931315 126931359 45 browser details YourSeq 33 131 166 2000 97.2% chr5 - 141607558 141607594 37 browser details YourSeq 32 126 158 2000 100.0% chr7 - 114055953 114055986 34 browser details YourSeq 32 119 166 2000 83.4% chr5 + 113031117 113031164 48 browser details YourSeq 27 110 144 2000 88.6% chr2 - 31224812 31224846 35 browser details YourSeq 27 129 164 2000 93.6% chr12 - 61517675 61517711 37 browser details YourSeq 27 343 389 2000 74.0% chrX + 64521917 64521962 46 browser details YourSeq 27 1499 1541 2000 86.3% chr18 + 70486109 70486149 41 browser details YourSeq 26 125 166 2000 81.0% chr3 - 122443701 122443742 42 browser details YourSeq 26 142 167 2000 100.0% chr2 - 101554676 101554701 26 browser details YourSeq 26 138 163 2000 100.0% chr12 - 23655161 23655186 26 browser details YourSeq 26 138 163 2000 100.0% chr12 - 24108901 24108926 26 browser details YourSeq 26 138 163 2000 100.0% chr12 - 22756611 22756636 26 Note: The 2000 bp section upstream of Exon 4 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% chr11 - 74433701 74435700 2000 browser details YourSeq 171 871 1058 2000 95.8% chr19 + 47944663 47944863 201 browser details YourSeq 165 867 1057 2000 94.2% chr17 - 45479885 45480298 414 browser details YourSeq 159 871 1058 2000 96.0% chr2 + 17538230 17538554 325 browser details YourSeq 151 920 1296 2000 94.1% chr7 + 34329217 34329799 583 browser details YourSeq 144 920 1293 2000 92.4% chr17 + 51839167 51945084 105918 browser details YourSeq 141 908 1074 2000 89.9% chr19 + 61127190 61127348 159 browser details YourSeq 140 928 1288 2000 86.4% chr11 + 95203485 95483406 279922 browser details YourSeq 139 920 1290 2000 86.1% chr11 - 94599846 94600157 312 browser details YourSeq 136 925 1319 2000 84.2% chr11 + 97741901 97742058 158 browser details YourSeq 134 908 1058 2000 94.7% chr17 - 84447097 84447263 167 browser details YourSeq 134 920 1291 2000 90.4% chr11 - 78961187 78961855 669 browser details YourSeq 129 919 1058 2000 94.2% chr19 - 11781112 11781248 137 browser details YourSeq 129 869 1058 2000 93.9% chr17 - 56275534 56275788 255 browser details YourSeq 129 920 1058 2000 95.0% chr3 + 108067932 108068069 138 browser details YourSeq 129 906 1053 2000 93.9% chr3 + 30751536 30751703 168 browser details YourSeq 128 920 1058 2000 93.4% chr8 - 105215608 105215743 136 browser details YourSeq 128 937 1231 2000 87.1% chr13 - 53273105 53273347 243 browser details YourSeq 128 920 1070 2000 90.4% chr16 + 4929727 4929873 147 browser details YourSeq 128 918 1058 2000 95.7% chr11 + 5388906 5389048 143 Note: The 2000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Rap1gap2 RAP1 GTPase activating protein 2 [ Mus musculus (house mouse) ] Gene ID: 380711, updated on 12-Aug-2019 Gene summary Official Symbol Rap1gap2 provided by MGI Official Full Name RAP1 GTPase activating protein 2 provided by MGI Primary source MGI:MGI:3028623 See related Ensembl:ENSMUSG00000038807 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 Garnl4; Gm1561; AU067654; mKIAA1039 Expression Broad expression in cortex adult (RPKM 24.4), frontal lobe adult (RPKM 21.6) and 23 other tissues See more Orthologs human all Genomic context Location: 11; 11 B5 See Rap1gap2 in Genome Data Viewer Exon count: 34 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (74383356..74610915, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (74196985..74403660, complement) Chromosome 11 - NC_000077.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Rap1gap2 ENSMUSG00000038807 Description RAP1 GTPase activating protein 2 [Source:MGI Symbol;Acc:MGI:3028623] Gene Synonyms Garnl4, LOC380710 Location Chromosome 11: 74,383,356-74,610,915 reverse strand. GRCm38:CM001004.2 About this gene This gene has 6 transcripts (splice variants), 291 orthologues, 6 paralogues, 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 Rap1gap2- ENSMUST00000102521.8 6565 712aa ENSMUSP00000099580.1 Protein coding CCDS25032 Q5SVL6 TSL:5 202 GENCODE basic APPRIS P2 Rap1gap2- ENSMUST00000238457.1 6389 772aa ENSMUSP00000158689.1 Protein coding - - GENCODE 206 basic APPRIS ALT2 Rap1gap2- ENSMUST00000208896.1 764 173aa ENSMUSP00000146728.1 Protein coding - A0A140LI95 CDS 3' 205 incomplete TSL:3 Rap1gap2- ENSMUST00000145524.1 450 90aa ENSMUSP00000117290.1 Protein coding - Q5SUE5 CDS 3' 204 incomplete TSL:5 Rap1gap2- ENSMUST00000047488.14 6382 758aa ENSMUSP00000040180.8 Nonsense mediated - A0A171EBK8 TSL:1 201 decay Rap1gap2- ENSMUST00000123440.1 5120 No - lncRNA - - TSL:1 203 protein Page 7 of 9 https://www.alphaknockout.com 247.56 kb Forward strand 74.4Mb 74.5Mb 74.6Mb Genes Ccdc92b-201 >protein coding (Comprehensive set... Contigs AL604065.7 > AL627348.12 > AL645971.12 > Genes (Comprehensive set... < Rap1gap2-202protein coding < Gm22993-201misc RNA < Rap1gap2-203lncRNA < Rap1gap2-205protein coding < Rap1gap2-206protein coding < Rap1gap2-201nonsense mediated decay < Rap1gap2-204protein coding Regulatory Build 74.4Mb 74.5Mb 74.6Mb Reverse strand 247.56 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000102521 < Rap1gap2-202protein coding Reverse strand 206.83 kb ENSMUSP00000099... MobiDB lite Low complexity (Seg) Superfamily Rap/Ran-GAP superfamily Pfam Rap GTPase activating protein domain PROSITE profiles Rap GTPase activating protein domain PANTHER PTHR15711 PTHR15711:SF17 Gene3D Rap/Ran-GAP superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 712 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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