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Mouse Myo18a Conditional Knockout Project (CRISPR/Cas9)

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Mouse Myo18a Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Myo18a Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Myo18a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Myo18a gene (NCBI Reference Sequence: NM_001291213 ; Ensembl: ENSMUSG00000000631 ) is located on Mouse chromosome 11. 41 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 41 (Transcript: ENSMUST00000168348). Exon 3~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Myo18a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-35M22 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: Homozygotes for a null allele exhibit embryonic lethality before E13.5, with abnormal myofibrils in cardiac myocytes. Exon 3 starts from about 18.31% of the coding region. The knockout of Exon 3~8 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1619 bp, and the size of intron 8 for 3'-loxP site insertion: 720 bp. The size of effective cKO region: ~2959 bp. The cKO 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 2 3 4 5 6 7 8 9 10 41 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Myo18a Homology arm cKO region loxP site Page 2 of 9 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(9459bp) | A(20.09% 1900) | C(26.37% 2494) | T(26.25% 2483) | G(27.3% 2582) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 9 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% chr11 + 77814370 77817369 3000 browser details YourSeq 65 1981 2085 3000 92.3% chr6 - 29964033 29964146 114 browser details YourSeq 62 1984 2081 3000 94.3% chr18 + 47218706 47218814 109 browser details YourSeq 62 1990 2078 3000 97.1% chr11 + 87006315 87006599 285 browser details YourSeq 62 1987 2085 3000 89.8% chr1 + 60310378 60310493 116 browser details YourSeq 59 1984 2083 3000 95.4% chr13 - 95447497 95447606 110 browser details YourSeq 59 1982 2078 3000 92.9% chr13 - 86162482 86162588 107 browser details YourSeq 58 1981 2060 3000 89.2% chr18 + 75749232 75749316 85 browser details YourSeq 57 1990 2078 3000 92.7% chr11 + 54407459 54407557 99 browser details YourSeq 55 1988 2085 3000 92.6% chr5 - 14882865 14883297 433 browser details YourSeq 55 1988 2079 3000 92.4% chr13 - 100659271 100659362 92 browser details YourSeq 53 1988 2078 3000 79.8% chr12 - 86293612 86293696 85 browser details YourSeq 53 2035 2152 3000 91.4% chr3 + 81090071 81090186 116 browser details YourSeq 50 1992 2063 3000 91.7% chr5 - 101582806 101582884 79 browser details YourSeq 50 1989 2083 3000 91.9% chr18 + 75409181 75409441 261 browser details YourSeq 50 1994 2076 3000 80.7% chr11 + 3981318 3981396 79 browser details YourSeq 49 1989 2082 3000 87.7% chr18 - 34917286 34917392 107 browser details YourSeq 48 1987 2086 3000 80.8% chr4 - 133555000 133555086 87 browser details YourSeq 45 1991 2064 3000 93.7% chr7 + 118379035 118379107 73 browser details YourSeq 43 2021 2085 3000 87.8% chr6 - 54724634 54724703 70 Note: The 3000 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 3000 1 3000 3000 100.0% chr11 + 77820329 77823328 3000 browser details YourSeq 90 1863 2061 3000 94.3% chr9 + 119637669 119638072 404 browser details YourSeq 68 1957 2091 3000 94.0% chr3 + 155825140 155825275 136 browser details YourSeq 65 1981 2064 3000 94.6% chr19 - 54194138 54194402 265 browser details YourSeq 52 1978 2055 3000 95.0% chr10 + 24202614 24202714 101 browser details YourSeq 45 2001 2049 3000 93.7% chr12 + 96121763 96121810 48 browser details YourSeq 35 2014 2048 3000 100.0% chr13 - 45813402 45813436 35 browser details YourSeq 29 1969 2004 3000 96.9% chr12 - 107858533 107858571 39 browser details YourSeq 25 1981 2005 3000 100.0% chr3 + 53674762 53674786 25 browser details YourSeq 24 2037 2060 3000 100.0% chr8 - 64575644 64575667 24 browser details YourSeq 22 2319 2340 3000 100.0% chr1 - 32110640 32110661 22 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 49644561 49644581 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 45128721 45128741 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 32800371 32800391 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 18430381 18430401 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 18775251 18775271 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 16145316 16145336 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 15115541 15115561 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 9140491 9140511 21 browser details YourSeq 21 2319 2339 3000 100.0% chr1 - 6885391 6885411 21 Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 4 of 9 https://www.alphaknockout.com Gene and protein information: Myo18a myosin XVIIIA [ Mus musculus (house mouse) ] Gene ID: 360013, updated on 29-Sep-2019 Gene summary Official Symbol Myo18a provided by MGI Official Full Name myosin XVIIIA provided by MGI Primary source MGI:MGI:2667185 See related Ensembl:ENSMUSG00000000631 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 MAJN; MyoPDZ; MysPDZ; SP-R210 Expression Ubiquitous expression in colon adult (RPKM 16.8), duodenum adult (RPKM 14.6) and 28 other tissues See more Orthologs human all Genomic context Location: 11; 11 B5 See Myo18a in Genome Data Viewer Exon count: 49 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (77763240..77865988) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (77590767..77679482) Chromosome 11 - NC_000077.6 Page 5 of 9 https://www.alphaknockout.com Transcript information: This gene has 24 transcripts Gene: Myo18a ENSMUSG00000000631 Description myosin XVIIIA [Source:MGI Symbol;Acc:MGI:2667185] Gene Synonyms MyoPDZ Location Chromosome 11: 77,763,246-77,865,980 forward strand. GRCm38:CM001004.2 About this gene This gene has 24 transcripts (splice variants), 261 orthologues, 42 paralogues, is a member of 1 Ensembl protein family and is associated with 17 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Myo18a-207 ENSMUST00000102488.7 7409 2035aa ENSMUSP00000099546.1 Protein coding CCDS25085 Q9JMH9 TSL:5 GENCODE basic APPRIS P3 Myo18a-203 ENSMUST00000092887.10 7304 2035aa ENSMUSP00000090563.4 Protein coding CCDS25085 Q9JMH9 TSL:1 GENCODE basic APPRIS P3 Myo18a-222 ENSMUST00000168348.7 6582 2083aa ENSMUSP00000130696.1 Protein coding CCDS70253 E9QAX2 TSL:1 GENCODE basic Myo18a-223 ENSMUST00000169105.7 6475 2047aa ENSMUSP00000132149.1 Protein coding CCDS70252 B2RRE2 TSL:1 GENCODE basic APPRIS ALT2 Myo18a-224 ENSMUST00000172303.9 6339 1722aa ENSMUSP00000129098.3 Protein coding CCDS83849 A0A1C7ZN10 TSL:1 GENCODE basic Myo18a-205 ENSMUST00000100794.9 6182 1700aa ENSMUSP00000098358.3 Protein coding CCDS78997 E9Q405 TSL:1 GENCODE basic Myo18a-208 ENSMUST00000108375.8 7513 2050aa ENSMUSP00000104012.2 Protein coding - Q9JMH9 TSL:5 GENCODE basic APPRIS ALT2 Myo18a-209 ENSMUST00000108376.8 7374 1998aa ENSMUSP00000104013.2 Protein coding - Q9JMH9 TSL:5 GENCODE basic Myo18a-201 ENSMUST00000000645.12 7307 2036aa ENSMUSP00000000645.6 Protein coding - K3W4L0 TSL:5 GENCODE basic Myo18a-211 ENSMUST00000130627.8 6202 2062aa ENSMUSP00000119839.2 Protein coding - Q9JMH9 TSL:5 GENCODE basic Myo18a-221 ENSMUST00000167856.7 6132 1642aa ENSMUSP00000128487.1 Protein coding - E9QA74 TSL:5 GENCODE basic Myo18a-210 ENSMUST00000130305.8 5450 1716aa ENSMUSP00000119574.2 Protein coding - Q9JMH9 TSL:5 GENCODE basic Myo18a-220 ENSMUST00000164334.7 5381 1719aa ENSMUSP00000131771.1 Protein coding - Q9JMH9 TSL:5 GENCODE basic Myo18a-202 ENSMUST00000092884.10 5117 1704aa ENSMUSP00000090560.4 Protein coding - Q9JMH9 TSL:5 GENCODE basic Myo18a-215 ENSMUST00000135375.3 2014 501aa ENSMUSP00000117044.3 Protein coding - F6ZGN3 CDS 5' incomplete TSL:5 Myo18a-217 ENSMUST00000151373.3 1076 307aa ENSMUSP00000123256.3
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