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Mouse Renbp Conditional Knockout Project (CRISPR/Cas9)

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Mouse Renbp Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Renbp Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Renbp conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Renbp gene (NCBI Reference Sequence: NM_023132 ; Ensembl: ENSMUSG00000031387 ) is located on Mouse chromosome X. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 11 (Transcript: ENSMUST00000116578). Exon 1~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Renbp gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-300A24 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 normal function of the renin-angiotensin system and blood pressure regulation; however, abnormalites in urine carbohydrates were noted. Exon 1~6 covers 53.49% of the coding region. Start codon is in exon 1, and stop codon is in exon 11. The size of intron 6 for 3'-loxP site insertion: 755 bp. The size of effective cKO region: ~1888 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele A T 5' G gRNA region 3' 1 2 3 4 5 6 7 8 11 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Renbp cKO region loxP site Page 2 of 8 https://www.alphaknockout.com 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(8128bp) | A(27.21% 2212) | C(23.7% 1926) | T(24.37% 1981) | G(24.72% 2009) 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. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chrX - 73930744 73933743 3000 browser details YourSeq 316 1968 2343 3000 94.2% chr1 - 58455897 58456278 382 browser details YourSeq 316 1968 2342 3000 94.9% chr1 - 17234579 17235041 463 browser details YourSeq 315 1962 2331 3000 94.9% chr10 + 77148867 77174100 25234 browser details YourSeq 310 1969 2362 3000 94.1% chr5 + 109947481 109948395 915 browser details YourSeq 307 1914 2329 3000 94.1% chr4 + 130690438 130690933 496 browser details YourSeq 306 1953 2331 3000 93.8% chr7 - 29499774 29500424 651 browser details YourSeq 304 1962 2317 3000 94.5% chr7 + 141397004 141397546 543 browser details YourSeq 302 1968 2331 3000 93.7% chr3 - 62947773 62948178 406 browser details YourSeq 299 1969 2329 3000 94.9% chr7 - 127720278 127720854 577 browser details YourSeq 296 1968 2317 3000 91.4% chr13 - 12678156 12678493 338 browser details YourSeq 292 1968 2331 3000 90.4% chr5 + 113997852 113998182 331 browser details YourSeq 289 1968 2317 3000 95.6% chr10 + 121651564 121651978 415 browser details YourSeq 274 1964 2317 3000 91.9% chr7 + 127518999 127519332 334 browser details YourSeq 271 1967 2311 3000 93.6% chr5 - 102507101 102507460 360 browser details YourSeq 234 2032 2317 3000 96.1% chr11 - 53369721 53370386 666 browser details YourSeq 227 2057 2331 3000 95.3% chr17 + 28993116 28994163 1048 browser details YourSeq 225 1964 2331 3000 93.8% chr11 - 23321380 23322057 678 browser details YourSeq 220 1702 2315 3000 87.7% chr11 + 76533559 76534042 484 browser details YourSeq 219 1712 2331 3000 85.4% chr10 - 62926070 62926647 578 Note: The 3000 bp section upstream of Exon 1 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% chrX - 73925866 73928865 3000 browser details YourSeq 409 306 2051 3000 92.6% chr16 - 32553568 33022366 468799 browser details YourSeq 324 1858 2351 3000 91.2% chr2 - 32075708 32373108 297401 browser details YourSeq 319 1252 2074 3000 94.2% chr18 - 36051643 36393937 342295 browser details YourSeq 304 1251 2052 3000 94.0% chr14 - 46820216 47388703 568488 browser details YourSeq 292 1867 2189 3000 96.6% chr17 + 6076449 6077073 625 browser details YourSeq 286 1806 2189 3000 89.9% chr13 - 73715381 73715708 328 browser details YourSeq 284 1870 2189 3000 95.3% chr2 + 73869768 73870106 339 browser details YourSeq 281 1878 2350 3000 92.8% chr11 - 104282953 104283506 554 browser details YourSeq 272 1805 2185 3000 92.8% chr19 + 8849730 8850143 414 browser details YourSeq 267 1867 2189 3000 93.2% chr15 - 102125654 102125978 325 browser details YourSeq 264 1867 2189 3000 92.7% chr17 + 28559191 28559722 532 browser details YourSeq 255 1809 2351 3000 85.9% chr11 - 119206063 119206505 443 browser details YourSeq 254 1867 2352 3000 86.4% chr12 + 118210740 118211096 357 browser details YourSeq 247 1867 2343 3000 85.5% chr1 + 120060145 120060512 368 browser details YourSeq 245 1867 2343 3000 89.4% chr12 - 76487588 76488045 458 browser details YourSeq 245 1867 2343 3000 92.8% chr5 + 135401862 135402439 578 browser details YourSeq 245 1867 2167 3000 93.3% chr15 + 80957808 80958255 448 browser details YourSeq 244 1870 2274 3000 88.6% chr11 - 30482470 30482833 364 browser details YourSeq 238 1867 2341 3000 84.9% chr10 - 121463104 121463516 413 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Renbp renin binding protein [ Mus musculus (house mouse) ] Gene ID: 19703, updated on 12-Aug-2019 Gene summary Official Symbol Renbp provided by MGI Official Full Name renin binding protein provided by MGI Primary source MGI:MGI:105940 See related Ensembl:ENSMUSG00000031387 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 Age; Rnbp Expression Biased expression in placenta adult (RPKM 61.2), kidney adult (RPKM 37.8) and 14 other tissues See more Orthologs human all Genomic context Location: X A7.3; X 37.49 cM See Renbp in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (73922121..73930850, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (71167460..71176189, complement) Chromosome X - NC_000086.7 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Renbp ENSMUSG00000031387 Description renin binding protein [Source:MGI Symbol;Acc:MGI:105940] Location Chromosome X: 73,922,121-73,930,850 reverse strand. GRCm38:CM001013.2 About this gene This gene has 10 transcripts (splice variants), 160 orthologues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Renbp- ENSMUST00000116578.7 1418 430aa ENSMUSP00000112277.1 Protein coding CCDS41015 P82343 TSL:1 202 GENCODE basic APPRIS P2 Renbp- ENSMUST00000114379.7 1318 416aa ENSMUSP00000110020.1 Protein coding CCDS57763 P82343 TSL:1 201 GENCODE basic Renbp- ENSMUST00000155597.1 1310 419aa ENSMUSP00000116549.1 Protein coding - D6RHA2 TSL:5 209 GENCODE basic APPRIS ALT2 Renbp- ENSMUST00000165359.2 514 48aa ENSMUSP00000132729.2 Nonsense mediated - G3XA46 TSL:3 210 decay Renbp- ENSMUST00000135711.7 2125 No - Retained intron - - TSL:1 204 protein Renbp- ENSMUST00000146371.7 1407 No - Retained intron - - TSL:1 206 protein Renbp- ENSMUST00000150241.1 490 No - Retained intron - - TSL:2 208 protein Renbp- ENSMUST00000135213.7 416 No - Retained intron - - TSL:2 203 protein Renbp- ENSMUST00000149130.1 349 No - Retained intron - - TSL:1 207 protein Renbp- ENSMUST00000144771.1 548 No - lncRNA - - TSL:5 205 protein Page 6 of 8 https://www.alphaknockout.com 28.73 kb Forward strand 73.92Mb 73.93Mb 73.94Mb Contigs AL672002.14 > Genes (Comprehensive set... < Arhgap4-209retained intron < Renbp-202protein coding < Naa10-201protein coding < Renbp-201protein coding < Naa10-208lncRNA < Naa10-211retained intron< Renbp-205lncRNA < Renbp-210nonsense mediated decay < Naa10-204protein coding < Renbp-203retained intron < Renbp-206retained intron < Naa10-203protein coding < Renbp-204retained intron < Naa10-206protein coding < Renbp-209protein coding < Naa10-205protein coding < Renbp-207retained intron < Naa10-209lncRNA < Naa10-212retained intron < Renbp-208retained intron < Naa10-202protein coding < Naa10-207retained intron < Naa10-210retained intron Regulatory Build 73.92Mb 73.93Mb 73.94Mb Reverse strand
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