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Mouse Arhgap27 Knockout Project (CRISPR/Cas9)

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Mouse Arhgap27 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Arhgap27 Knockout Project (CRISPR/Cas9) Objective: To create a Arhgap27 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Arhgap27 gene (NCBI Reference Sequence: NM_001205236.1 ; Ensembl: ENSMUSG00000034255 ) is located on Mouse chromosome 11. 16 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000107024). Exon 4~10 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: Exon 4 starts from about 48.25% of the coding region. Exon 4~10 covers 24.32% of the coding region. The size of effective KO region: ~2282 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 9 10 11 16 Legends Exon of mouse Arhgap27 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 178 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 124 bp section downstream of Exon 10 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(178bp) | A(21.91% 39) | C(36.52% 65) | T(23.03% 41) | G(18.54% 33) Note: The 178 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(124bp) | A(19.35% 24) | C(23.39% 29) | T(20.16% 25) | G(37.1% 46) Note: The 124 bp section downstream of Exon 10 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 178 1 178 178 100.0% chr11 - 103339277 103339454 178 browser details YourSeq 22 39 61 178 100.0% chr1_GL456213_random + 35506 35531 26 Note: The 178 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 124 1 124 124 100.0% chr11 - 103334134 103334257 124 Note: The 124 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Arhgap27 Rho GTPase activating protein 27 [ Mus musculus (house mouse) ] Gene ID: 544817, updated on 26-Jun-2020 Gene summary Official Symbol Arhgap27 provided by MGI Official Full Name Rho GTPase activating protein 27 provided by MGI Primary source MGI:MGI:1916903 See related Ensembl:ENSMUSG00000034255 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 Sh3d20; Gm21552; 2310069I04Rik; 5730442P18Rik Expression Broad expression in colon adult (RPKM 40.0), duodenum adult (RPKM 33.5) and 18 other tissues See more Orthologs human all Genomic context Location: 11; 11 E1 See Arhgap27 in Genome Data Viewer Exon count: 20 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (103331484..103363692, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (103192814..103206016, complement) Chromosome 11 - NC_000077.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Arhgap27 ENSMUSG00000034255 Description Rho GTPase activating protein 27 [Source:MGI Symbol;Acc:MGI:1916903] Gene Synonyms 2310069I04Rik, 5730442P18Rik, Sh3d20 Location Chromosome 11: 103,331,497-103,363,692 reverse strand. GRCm38:CM001004.2 About this gene This gene has 11 transcripts (splice variants), 356 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Arhgap27- ENSMUST00000107024.7 3976 869aa ENSMUSP00000102639.1 Protein coding CCDS56815 A2AB59 TSL:5 204 GENCODE basic APPRIS ALT2 Arhgap27- ENSMUST00000041385.13 3512 670aa ENSMUSP00000039427.7 Protein coding CCDS25517 A2AB59 TSL:1 201 GENCODE basic APPRIS P3 Arhgap27- ENSMUST00000092557.5 1504 243aa ENSMUSP00000102637.2 Protein coding CCDS36350 A2AKN5 TSL:2 202 GENCODE basic Arhgap27- ENSMUST00000136491.2 748 91aa ENSMUSP00000128051.1 Protein coding - E9Q6V8 CDS 3' 207 incomplete TSL:3 Arhgap27- ENSMUST00000107023.2 379 123aa ENSMUSP00000102638.2 Protein coding - A2AB70 CDS 3' 203 incomplete TSL:2 Arhgap27- ENSMUST00000163250.1 114 No - Processed - - TSL:1 211 protein transcript Arhgap27- ENSMUST00000116102.8 4835 No - Retained intron - - TSL:2 205 protein Arhgap27- ENSMUST00000150122.1 857 No - Retained intron - - TSL:3 210 protein Arhgap27- ENSMUST00000139830.7 726 No - Retained intron - - TSL:5 208 protein Arhgap27- ENSMUST00000124490.1 673 No - Retained intron - - TSL:3 206 protein Arhgap27- ENSMUST00000144194.1 434 No - Retained intron - - TSL:3 209 protein Page 7 of 9 https://www.alphaknockout.com 52.20 kb Forward strand 103.33Mb 103.34Mb 103.35Mb 103.36Mb 103.37Mb Genes Arhgap27os3-201 >antisense Arhgap27os2-201 >antisense (Comprehensive set... Arhgap27os1-201 >antisense Contigs AL662804.18 > AL772325.4 > Genes < Arhgap27-204protein coding < Plekhm1-202nonsense mediated decay (Comprehensive set... < Arhgap27-205retained intron < Plekhm1-201protein coding < Arhgap27-201protein coding < Arhgap27-202protein coding < Arhgap27-210retained intron < Arhgap27-211processed transcript < Arhgap27-209retained intron < Arhgap27-208retained intron < Arhgap27-206retained intron < Arhgap27-207protein coding < Arhgap27-203protein coding Regulatory Build 103.33Mb 103.34Mb 103.35Mb 103.36Mb 103.37Mb Reverse strand 52.20 kb Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000107024 < Arhgap27-204protein coding Reverse strand 29.40 kb ENSMUSP00000102... MobiDB lite Low complexity (Seg) Superfamily SH3-like domain superfamily WW domain superfamily SSF50729 Rho GTPase activation protein Quinoprotein alcohol dehydrogenase-like superfamily SMART WW domain Pleckstrin homology domain Rho GTPase-activating protein domain Pfam WW domain Pleckstrin homology domain Rho GTPase-activating protein domain PROSITE profiles SH3 domain WW domain Pleckstrin homology domain Rho GTPase-activating protein domain PANTHER PTHR23176:SF104 PTHR23176 Gene3D 2.30.30.40 2.20.70.10 PH-like domain superfamily Rho GTPase activation protein CDD cd12069 WW domain cd13233 cd04403 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 869 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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