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Mouse Rpa3 Knockout Project (CRISPR/Cas9)

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Mouse Rpa3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rpa3 Knockout Project (CRISPR/Cas9) Objective: To create a Rpa3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rpa3 gene (NCBI Reference Sequence: NM_026632.4 ; Ensembl: ENSMUSG00000012483 ) is located on Mouse chromosome 6. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000012627). Exon 3 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 3 starts from about 48.21% of the coding region. Exon 3 covers 30.03% of the coding region. The size of effective KO region: ~109 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 Legends Exon of mouse Rpa3 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 883 bp section upstream of Exon 3 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 447 bp section downstream 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(883bp) | A(31.82% 281) | C(15.18% 134) | T(38.62% 341) | G(14.38% 127) Note: The 883 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(447bp) | A(36.24% 162) | C(14.54% 65) | T(31.99% 143) | G(17.23% 77) Note: The 447 bp section downstream 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. Page 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 883 1 883 883 100.0% chr6 - 8256804 8257686 883 browser details YourSeq 52 373 430 883 96.5% chr5 + 123843564 123843637 74 browser details YourSeq 46 370 417 883 98.0% chr1 - 110871050 110871097 48 browser details YourSeq 44 377 421 883 100.0% chr11 + 68125874 68125920 47 browser details YourSeq 43 378 420 883 100.0% chr18 - 27056625 27056667 43 browser details YourSeq 42 368 411 883 97.8% chr1 - 30767076 30767119 44 browser details YourSeq 40 380 421 883 100.0% chr1 - 100623304 100623349 46 browser details YourSeq 28 373 420 883 84.4% chr11 + 7742186 7742231 46 browser details YourSeq 28 225 260 883 75.9% chr1 + 54341482 54341510 29 browser details YourSeq 26 426 451 883 100.0% chr2 - 78779210 78779235 26 browser details YourSeq 26 399 424 883 100.0% chr1 + 3403481 3403506 26 browser details YourSeq 25 301 331 883 85.2% chr6 - 122590955 122590983 29 browser details YourSeq 21 740 760 883 100.0% chr6 - 129353471 129353491 21 browser details YourSeq 21 218 238 883 100.0% chr9 + 52392811 52392831 21 browser details YourSeq 20 660 679 883 100.0% chr7 - 97093602 97093621 20 Note: The 883 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 447 1 447 447 100.0% chr6 - 8256248 8256694 447 browser details YourSeq 32 56 104 447 94.5% chr1 - 176120825 176120874 50 browser details YourSeq 27 196 224 447 100.0% chr19 - 39403974 39404005 32 browser details YourSeq 26 148 184 447 96.5% chr1 + 23076505 23076542 38 browser details YourSeq 25 189 217 447 88.9% chr11 + 25048799 25048826 28 browser details YourSeq 24 199 222 447 100.0% chr10 - 18279293 18279316 24 browser details YourSeq 23 34 59 447 96.2% chr11 - 85100356 85100392 37 browser details YourSeq 22 281 305 447 95.9% chr2 - 155342616 155342641 26 browser details YourSeq 22 420 443 447 95.9% chr10 - 62990265 62990288 24 browser details YourSeq 22 202 226 447 95.9% chr1 + 150329371 150329396 26 browser details YourSeq 21 291 311 447 100.0% chr3 - 69050576 69050596 21 Note: The 447 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Rpa3 replication protein A3 [ Mus musculus (house mouse) ] Gene ID: 68240, updated on 26-Jun-2020 Gene summary Official Symbol Rpa3 provided by MGI Official Full Name replication protein A3 provided by MGI Primary source MGI:MGI:1915490 See related Ensembl:ENSMUSG00000012483 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 14kDa; C330026P08Rik Expression Biased expression in CNS E11.5 (RPKM 31.8), liver E14 (RPKM 27.7) and 13 other tissues See more Orthologs human all Genomic context Location: 6; 6 A1 See Rpa3 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (8255936..8259141, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (8205936..8209141, complement) Chromosome 6 - NC_000072.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Rpa3 ENSMUSG00000012483 Description replication protein A3 [Source:MGI Symbol;Acc:MGI:1915490] Gene Synonyms 14kDa, C330026P08Rik Location Chromosome 6: 8,255,936-8,259,173 reverse strand. GRCm38:CM000999.2 About this gene This gene has 2 transcripts (splice variants), 277 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rpa3-201 ENSMUST00000012627.4 670 121aa ENSMUSP00000012627.4 Protein coding CCDS19909 Q9CQ71 TSL:1 GENCODE basic APPRIS P1 Rpa3-202 ENSMUST00000159594.1 682 No protein - Retained intron - - TSL:2 23.24 kb Forward strand 8.250Mb 8.255Mb 8.260Mb 8.265Mb Genes Umad1-202 >protein coding (Comprehensive set... Umad1-203 >protein coding Umad1-208 >protein coding Umad1-206 >protein coding Umad1-204 >protein coding Umad1-207 >processed transcript Gm45062-201 >nonsense mediated decay Contigs < AC158661.7 Genes (Comprehensive set... < Rpa3-201protein coding < Rpa3-202retained intron Regulatory Build 8.250Mb 8.255Mb 8.260Mb 8.265Mb Reverse strand 23.24 kb Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Regulation Legend CTCF Promoter Promoter Flank Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000012627 < Rpa3-201protein coding Reverse strand 3.24 kb ENSMUSP00000012... Superfamily Nucleic acid-binding, OB-fold Pfam Replication factor A protein 3 PANTHER PTHR15114 Gene3D 2.40.50.140 CDD cd04479 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant Scale bar 0 20 40 60 80 100 121 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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