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Mouse Reps2 Knockout Project (CRISPR/Cas9)

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Mouse Reps2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Reps2 Knockout Project (CRISPR/Cas9) Objective: To create a Reps2 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Reps2 gene (NCBI Reference Sequence: NM_178256 ; Ensembl: ENSMUSG00000040855 ) is located on Mouse chromosome X. 18 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000101102). Exon 2 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 2 starts from about 12.09% of the coding region. Exon 2 covers 6.38% of the coding region. The size of effective KO region: ~124 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 gRNA region 5' gRNA region 3' 1 2 18 Legends Exon of mouse Reps2 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 2000 bp section upstream of Exon 2 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 2000 bp section downstream of Exon 2 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(2000bp) | A(24.25% 485) | C(19.25% 385) | T(33.35% 667) | G(23.15% 463) Note: The 2000 bp section upstream of Exon 2 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(24.55% 491) | C(21.05% 421) | T(33.35% 667) | G(21.05% 421) Note: The 2000 bp section downstream of Exon 2 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 2000 1 2000 2000 100.0% chrX - 162565352 162567351 2000 browser details YourSeq 496 23 536 2000 98.3% chr9 + 19927810 19928323 514 browser details YourSeq 491 40 541 2000 99.3% chr8 + 70508263 70510372 2110 browser details YourSeq 491 21 530 2000 97.6% chr4 + 3973096 3973595 500 browser details YourSeq 488 25 536 2000 96.9% chr9 + 72174495 72175000 506 browser details YourSeq 454 21 536 2000 94.2% chr18 - 35536542 35537039 498 browser details YourSeq 407 37 501 2000 93.8% chr11 + 96795367 96795831 465 browser details YourSeq 364 35 508 2000 88.2% chr5 + 29719258 29719719 462 browser details YourSeq 361 33 508 2000 88.8% chr11 + 60610068 60667078 57011 browser details YourSeq 361 34 508 2000 87.3% chr11 + 60679639 60680101 463 browser details YourSeq 358 33 508 2000 86.8% chr11 + 60619278 60619740 463 browser details YourSeq 358 47 508 2000 87.9% chr11 + 60628522 60628976 455 browser details YourSeq 350 47 508 2000 87.2% chr11 + 60560864 60561318 455 browser details YourSeq 349 34 508 2000 88.2% chr11 + 60651003 60693088 42086 browser details YourSeq 329 55 538 2000 85.1% chrX - 153166612 153167078 467 browser details YourSeq 329 21 512 2000 88.8% chr3 + 95963933 95964405 473 browser details YourSeq 303 111 498 2000 88.0% chr11 + 60641863 60642245 383 browser details YourSeq 301 111 498 2000 89.5% chr11 + 60571625 60610520 38896 browser details YourSeq 301 111 498 2000 87.8% chr11 + 60600929 60601311 383 browser details YourSeq 301 111 498 2000 87.8% chr11 + 60591723 60592105 383 Note: The 2000 bp section upstream of Exon 2 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% chrX - 162563228 162565227 2000 browser details YourSeq 441 1490 2000 2000 94.4% chr18 + 30545894 30639988 94095 browser details YourSeq 439 1493 2000 2000 94.1% chr13 - 24118738 24119259 522 browser details YourSeq 438 1493 1999 2000 93.9% chr4 - 100082341 100082861 521 browser details YourSeq 438 1503 1999 2000 94.8% chr10 - 122431905 122432411 507 browser details YourSeq 436 1493 1998 2000 93.8% chrX + 61455847 61456372 526 browser details YourSeq 436 1487 1999 2000 93.7% chr11 + 21649144 21649677 534 browser details YourSeq 435 1493 1999 2000 94.2% chr13 - 96441789 96442306 518 browser details YourSeq 435 1493 2000 2000 93.8% chr18 + 3727296 3727821 526 browser details YourSeq 433 1497 2000 2000 94.0% chrX - 75718603 75719219 617 browser details YourSeq 433 1321 1999 2000 90.6% chr14 - 45097495 45098012 518 browser details YourSeq 432 1493 2000 2000 92.9% chr5 - 62834369 62834889 521 browser details YourSeq 432 1493 2000 2000 93.1% chr15 + 33538798 33539319 522 browser details YourSeq 430 1490 2000 2000 93.6% chr19 - 49554877 49555388 512 browser details YourSeq 430 1505 2000 2000 93.9% chr7 + 132590171 132590679 509 browser details YourSeq 430 1496 1999 2000 93.6% chr14 + 13131555 13132068 514 browser details YourSeq 427 1493 2000 2000 93.0% chr2 + 83779651 83780166 516 browser details YourSeq 427 1490 2000 2000 93.5% chr2 + 67237114 67237621 508 browser details YourSeq 426 1491 1999 2000 93.6% chr8 - 39928605 39929126 522 browser details YourSeq 424 1494 2000 2000 93.0% chr11 - 42343332 42343848 517 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Reps2 RALBP1 associated Eps domain containing protein 2 [ Mus musculus (house mouse) ] Gene ID: 194590, updated on 7-Oct-2019 Gene summary Official Symbol Reps2 provided by MGI Official Full Name RALBP1 associated Eps domain containing protein 2 provided by MGI Primary source MGI:MGI:2663511 See related Ensembl:ENSMUSG00000040855 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 POB1 Expression Broad expression in cortex adult (RPKM 8.4), frontal lobe adult (RPKM 7.3) and 21 other tissues See more Orthologs human all Genomic context Location: X; X F4 See Reps2 in Genome Data Viewer Exon count: 20 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (162411952..162643705, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (158849886..159081533, complement) Chromosome X - NC_000086.7 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Reps2 ENSMUSG00000040855 Description RALBP1 associated Eps domain containing protein 2 [Source:MGI Symbol;Acc:MGI:2663511] Gene Synonyms POB1 Location Chromosome X: 162,411,954-162,643,649 reverse strand. GRCm38:CM001013.2 About this gene This gene has 5 transcripts (splice variants), 194 orthologues, 10 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Reps2-202 ENSMUST00000112334.7 7675 647aa ENSMUSP00000107953.1 Protein coding CCDS72462 A2AFI8 TSL:1 GENCODE basic APPRIS ALT2 Reps2-201 ENSMUST00000101102.1 7630 648aa ENSMUSP00000098661.1 Protein coding CCDS30508 B9EI38 TSL:1 GENCODE basic APPRIS P3 Reps2-204 ENSMUST00000155043.1 667 No protein - Retained intron - - TSL:3 Reps2-203 ENSMUST00000154424.7 7146 No protein - lncRNA - - TSL:1 Reps2-205 ENSMUST00000155863.1 554 No protein - lncRNA - - TSL:5 251.70 kb Forward strand 162.45Mb 162.50Mb 162.55Mb 162.60Mb 162.65Mb Genes Gm7331-201 >processed pseudogene (Comprehensive set... Contigs AL672039.9 > AL672123.16 > Genes < Reps2-201protein coding (Comprehensive set... < Reps2-202protein coding < Reps2-203lncRNA < Gm15206-201processed pseudogene < Reps2-205lncRNA < Reps2-204retained intron Regulatory Build 162.45Mb 162.50Mb 162.55Mb 162.60Mb 162.65Mb Reverse strand 251.70 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding processed transcript RNA gene pseudogene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000101102 < Reps2-201protein coding Reverse strand 231.65 kb ENSMUSP00000098... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily EF-hand domain pair SMART EH domain Pfam EH domain PROSITE profiles EH domain EF-hand domain PROSITE patterns EF-Hand 1, calcium-binding site PANTHER PTHR11216 PTHR11216:SF64 Gene3D 1.10.238.10 CDD EH domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 648 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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