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Mouse Atg10 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Atg10 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Atg10 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Atg10 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Atg10 gene (NCBI Reference Sequence: NM_025770 ; Ensembl: ENSMUSG00000021619 ) is located on Mouse chromosome 13. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000022119). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Atg10 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-326J5 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: Exon 2 starts from about 100% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 15505 bp, and the size of intron 2 for 3'-loxP site insertion: 54005 bp. The size of effective cKO region: ~605 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 8 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Atg10 Homology arm cKO region loxP site Page 2 of 7 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7105bp) | A(25.02% 1778) | C(20.99% 1491) | T(33.13% 2354) | G(20.86% 1482) 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 7 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% chr13 - 91208673 91211672 3000 browser details YourSeq 51 1015 1290 3000 64.8% chr1 + 59663951 59664067 117 browser details YourSeq 45 2206 2254 3000 96.0% chr13 - 91210904 91210952 49 browser details YourSeq 45 721 769 3000 96.0% chr13 - 91209419 91209467 49 browser details YourSeq 44 1016 1270 3000 62.5% chr16 - 73896853 73896946 94 browser details YourSeq 43 1052 1290 3000 60.7% chr17 + 20626849 20626927 79 browser details YourSeq 39 1015 1259 3000 59.2% chr15 - 62186117 62186201 85 browser details YourSeq 39 1055 1274 3000 59.2% chr14 - 113721445 113721504 60 browser details YourSeq 39 1457 1533 3000 89.8% chr17 + 57193134 57193219 86 browser details YourSeq 34 1244 1293 3000 84.0% chr14 + 20328277 20328326 50 browser details YourSeq 34 1244 1293 3000 84.0% chr12 + 94095567 94095616 50 browser details YourSeq 33 1464 1526 3000 76.2% chr1 + 193411511 193411573 63 browser details YourSeq 31 1236 1274 3000 89.8% chr13 - 103611365 103611403 39 browser details YourSeq 31 1055 1135 3000 75.7% chr1 - 21477381 21477454 74 browser details YourSeq 31 1257 1287 3000 100.0% chr4 + 117771990 117772020 31 browser details YourSeq 31 1085 1135 3000 84.7% chr13 + 93378882 93378931 50 browser details YourSeq 30 1466 1523 3000 75.9% chr18 - 15017809 15017866 58 browser details YourSeq 30 1244 1275 3000 96.9% chr1 + 59588063 59588094 32 browser details YourSeq 29 1110 1148 3000 87.2% chr11 - 69508635 69508673 39 browser details YourSeq 28 1244 1275 3000 93.8% chr12 - 94944876 94944907 32 Note: The 3000 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 3000 1 3000 3000 100.0% chr13 - 91205068 91208067 3000 browser details YourSeq 318 606 1042 3000 89.2% chr17 + 74963682 74964122 441 browser details YourSeq 308 604 1042 3000 88.6% chr10 + 90113291 90113754 464 browser details YourSeq 303 597 1042 3000 88.4% chr6 + 14473151 14473609 459 browser details YourSeq 301 618 1042 3000 88.1% chr8 + 80209345 80209769 425 browser details YourSeq 300 600 1026 3000 88.2% chr19 + 49446849 49447276 428 browser details YourSeq 298 605 1044 3000 87.0% chr5 - 101735410 101735849 440 browser details YourSeq 293 573 1042 3000 84.3% chrX + 144263967 144264411 445 browser details YourSeq 293 595 1042 3000 89.7% chr15 + 92758302 92758767 466 browser details YourSeq 291 563 1039 3000 86.2% chr7 - 105837836 105838302 467 browser details YourSeq 290 605 1042 3000 88.0% chr16 - 64246766 64669994 423229 browser details YourSeq 290 618 1042 3000 84.6% chrX + 103147255 103147665 411 browser details YourSeq 290 597 1042 3000 87.0% chr5 + 89286998 89287446 449 browser details YourSeq 290 617 1042 3000 87.4% chr19 + 21481776 21482227 452 browser details YourSeq 287 654 1046 3000 89.8% chr7 - 102635875 102636269 395 browser details YourSeq 286 575 1042 3000 86.6% chrX - 139408150 139408585 436 browser details YourSeq 286 619 1042 3000 88.6% chr1 + 11283512 11283915 404 browser details YourSeq 285 618 1040 3000 84.8% chr3 - 69838701 69839129 429 browser details YourSeq 283 605 1042 3000 87.2% chrX + 164464564 164465002 439 browser details YourSeq 282 603 1038 3000 86.2% chrX - 28584265 28584728 464 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Atg10 autophagy related 10 [ Mus musculus (house mouse) ] Gene ID: 66795, updated on 10-Oct-2019 Gene summary Official Symbol Atg10 provided by MGI Official Full Name autophagy related 10 provided by MGI Primary source MGI:MGI:1914045 See related Ensembl:ENSMUSG00000021619 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 APG10; Agp10; Apg10l; Apg10p; Atg10l; AI852123; 5330424L23Rik; 5430428K15Rik Expression Ubiquitous expression in bladder adult (RPKM 1.2), heart adult (RPKM 0.9) and 25 other tissues See more Orthologs human all Genomic context Location: 13; 13 C3 See Atg10 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (90935348..91224725, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (91074954..91363592, complement) Chromosome 13 - NC_000079.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Atg10 ENSMUSG00000021619 Description autophagy related 10 [Source:MGI Symbol;Acc:MGI:1914045] Gene Synonyms 5330424L23Rik, 5430428K15Rik, APG10, Apg10l, Apg10p Location Chromosome 13: 90,935,356-91,223,968 reverse strand. GRCm38:CM001006.2 About this gene This gene has 3 transcripts (splice variants), 189 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Atg10- ENSMUST00000022119.5 1583 211aa ENSMUSP00000022119.4 Protein coding CCDS26675 A0A0R4J029 TSL:1 201 GENCODE basic APPRIS P1 Atg10- ENSMUST00000224449.1 922 71aa ENSMUSP00000153331.1 Nonsense mediated - A0A286YD93 - 203 decay Atg10- ENSMUST00000223729.1 826 No - lncRNA - - - 202 protein 308.61 kb Forward strand 91.0Mb 91.1Mb 91.2Mb Genes Gm17450-201 >pseudogene (Comprehensive set... Contigs AC108947.5 > < AC134461.4 < AC160980.5 Genes < Atg10-201protein coding (Comprehensive set... < Atg10-203nonsense mediated decay < Atg10-202lncRNA Regulatory Build 91.0Mb 91.1Mb 91.2Mb Reverse strand 308.61 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript pseudogene RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000022119 < Atg10-201protein coding Reverse strand 288.61 kb ENSMUSP00000022... Low complexity (Seg) Pfam Autophagy-related protein 3 PANTHER PTHR14957 Gene3D 3.30.1460.50 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained inframe insertion missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 211 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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