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Mouse Piezo2 Knockout Project (CRISPR/Cas9)

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Mouse Piezo2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Piezo2 Knockout Project (CRISPR/Cas9) Objective: To create a Piezo2 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Piezo2 gene (NCBI Reference Sequence: NM_001039485 ; Ensembl: ENSMUSG00000041482 ) is located on Mouse chromosome 18. 54 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 54 (Transcript: ENSMUST00000047480). Exon 6~7 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: Mice homozygous for a null allele exhibit impaired injury-induced tactile pain detection.Since the null allele lead to embronic lethalty, we highly recommond you thinking of generation a conditional knockout model. You can get the KO type by crossing with a deleter mice. Exon 6 starts from about 5.82% of the coding region. Exon 6~7 covers 5.02% of the coding region. The size of effective KO region: ~2092 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 6 7 54 Legends Exon of mouse Piezo2 Knockout region Page 2 of 9 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 6 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 7 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(29.65% 593) | C(21.8% 436) | T(28.05% 561) | G(20.5% 410) Note: The 2000 bp section upstream of Exon 6 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(32.05% 641) | C(19.55% 391) | T(25.6% 512) | G(22.8% 456) Note: The 2000 bp section downstream of Exon 7 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 9 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% chr18 - 63147007 63149006 2000 browser details YourSeq 148 191 375 2000 93.6% chr13 + 100480216 100680403 200188 browser details YourSeq 61 166 241 2000 94.3% chr12 - 80920937 80921026 90 browser details YourSeq 61 138 231 2000 84.9% chr4 + 155794593 155794678 86 browser details YourSeq 61 133 232 2000 95.5% chr2 + 167494195 167494515 321 browser details YourSeq 61 129 231 2000 95.6% chr11 + 121567552 121567828 277 browser details YourSeq 60 166 242 2000 94.1% chr18 + 56567416 56567510 95 browser details YourSeq 59 166 241 2000 94.0% chr12 - 72695585 72695685 101 browser details YourSeq 59 167 241 2000 92.7% chr2 + 5057924 5058023 100 browser details YourSeq 58 178 242 2000 96.8% chr4 - 144070322 144070397 76 browser details YourSeq 58 166 240 2000 92.6% chr17 - 88425912 88426006 95 browser details YourSeq 58 166 240 2000 95.4% chr16 - 50506752 50506847 96 browser details YourSeq 58 166 240 2000 93.9% chr2 + 181704540 181704641 102 browser details YourSeq 58 166 240 2000 93.9% chr11 + 54945600 54945697 98 browser details YourSeq 57 166 240 2000 92.5% chr17 - 26081264 26081346 83 browser details YourSeq 57 166 322 2000 76.2% chr16 + 21703931 21704024 94 browser details YourSeq 56 166 239 2000 92.6% chr14 - 70307258 70307361 104 browser details YourSeq 56 166 240 2000 93.8% chr12 - 112864119 112864216 98 browser details YourSeq 56 166 236 2000 93.7% chr12 - 83835948 83836026 79 browser details YourSeq 56 176 240 2000 95.3% chr11 - 103012020 103012099 80 Note: The 2000 bp section upstream of Exon 6 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% chr18 - 63142915 63144914 2000 browser details YourSeq 151 1357 1531 2000 96.4% chr4 + 56920900 56921524 625 browser details YourSeq 137 1389 1537 2000 96.6% chr19 - 21264166 21264321 156 browser details YourSeq 135 1389 1536 2000 96.6% chr1 - 16658388 16658543 156 browser details YourSeq 133 1386 1532 2000 96.6% chr7 - 29203512 29272678 69167 browser details YourSeq 133 1386 1538 2000 95.3% chr1 - 63639395 63639558 164 browser details YourSeq 132 1389 1537 2000 95.2% chr10 - 68262810 68262964 155 browser details YourSeq 132 1386 1537 2000 95.4% chr5 + 144399275 144399440 166 browser details YourSeq 132 1389 1536 2000 96.6% chr2 + 155813526 155813686 161 browser details YourSeq 132 1391 1536 2000 96.6% chr1 + 136679735 136679900 166 browser details YourSeq 131 1389 1535 2000 95.2% chr18 + 67637986 67638144 159 browser details YourSeq 130 1386 1536 2000 94.1% chr8 - 25506305 25506473 169 browser details YourSeq 130 1385 1537 2000 93.5% chr10 - 127523254 127523421 168 browser details YourSeq 130 1392 1535 2000 95.9% chr1 - 36948569 36948727 159 browser details YourSeq 130 1386 1536 2000 93.3% chr2 + 157200425 157200588 164 browser details YourSeq 130 1389 1526 2000 97.9% chr2 + 16452010 16452177 168 browser details YourSeq 130 1389 1535 2000 95.9% chr1 + 155807416 155807577 162 browser details YourSeq 130 1390 1534 2000 96.5% chr1 + 153548657 153548813 157 browser details YourSeq 129 1389 1537 2000 95.8% chr15 - 79963522 79963680 159 browser details YourSeq 129 1256 1520 2000 85.6% chr10 - 7020294 7020459 166 Note: The 2000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Piezo2 piezo-type mechanosensitive ion channel component 2 [ Mus musculus (house mouse) ] Gene ID: 667742, updated on 14-Oct-2019 Gene summary Official Symbol Piezo2 provided by MGI Official Full Name piezo-type mechanosensitive ion channel component 2 provided by MGI Primary source MGI:MGI:1918781 See related Ensembl:ENSMUSG00000041482 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 Fam38b; Fam38b2; 5930434P17; 9030411M15Rik; 9430028L06Rik Expression Biased expression in limb E14.5 (RPKM 12.4), lung adult (RPKM 5.9) and 9 other tissuesS ee more Orthologs human all Genomic context Location: 18; 18 E1 See Piezo2 in Genome Data Viewer Exon count: 55 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (63010213..63387716, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (63169867..63546837, complement) Chromosome 18 - NC_000084.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 15 transcripts Gene: Piezo2 ENSMUSG00000041482 Description piezo-type mechanosensitive ion channel component 2 [Source:MGI Symbol;Acc:MGI:1918781] Gene Synonyms 9030411M15Rik, 9430028L06Rik, Fam38b, Fam38b2, Piezo2 Location Chromosome 18: 63,010,213-63,387,183 reverse strand. GRCm38:CM001011.2 About this gene This gene has 15 transcripts (splice variants), 283 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Piezo2- ENSMUST00000047480.12 10724 2824aa ENSMUSP00000040019.5 Protein coding CCDS29296 E9QNW4 TSL:1 202 GENCODE basic APPRIS P2 Piezo2- ENSMUST00000238156.1 8773 2690aa ENSMUSP00000157601.1 Protein coding - A0A494B9D1 GENCODE 215 basic Piezo2- ENSMUST00000183217.8 8564 2753aa ENSMUSP00000138758.2 Protein coding - S4R2S0 TSL:5 209 GENCODE basic APPRIS ALT2 Piezo2- ENSMUST00000238051.1 8337 2778aa ENSMUSP00000157827.1 Protein coding - A0A494B9V6 GENCODE 214 basic APPRIS ALT2 Piezo2- ENSMUST00000182166.8 1988 644aa ENSMUSP00000138754.2 Protein coding - Q8CD54 TSL:5 206 S4R2R6 GENCODE basic Piezo2- ENSMUST00000132576.8 1318 378aa ENSMUSP00000157610.1 Protein coding - A0A494B9E4 CDS 5' 204 incomplete TSL:1 Piezo2- ENSMUST00000046860.6 1125 375aa ENSMUSP00000036099.6 Protein coding - - CDS 5' and 3' 201 incomplete TSL:5 Piezo2- ENSMUST00000235366.1 689 230aa ENSMUSP00000158292.1 Protein coding - A0A494BB29 CDS 5' and 3' 210 incomplete Piezo2- ENSMUST00000137141.3 3200 409aa ENSMUSP00000117107.2 Nonsense mediated - F6YYK4 CDS 5' 205 decay incomplete TSL:1 Piezo2- ENSMUST00000123322.1 3739 No - Retained intron - - TSL:1 203 protein Piezo2- ENSMUST00000182177.2 2963 No - Retained intron - - TSL:1 207 protein Piezo2- ENSMUST00000237812.1 1803 No - Retained intron - - - 213 protein Piezo2- ENSMUST00000236438.1 1452 No - Retained intron - - - 212 protein Piezo2- ENSMUST00000235659.1 889 No - Retained intron - - - 211 protein Piezo2- ENSMUST00000182233.8 3459 No - lncRNA - - TSL:5 208 protein Page 7 of 9 https://www.alphaknockout.com 396.97 kb Forward strand 63.1Mb 63.2Mb 63.3Mb Genes Gm50172-201 >TEC (Comprehensive set... Contigs AC140554.3 > < AC132378.3 AC124391.3 > Genes < Piezo2-202protein coding (Comprehensive set..
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