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Mouse Piwil1 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Piwil1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Piwil1 gene (NCBI Reference Sequence: NM_021311 ; Ensembl: ENSMUSG00000029423 ) is located on Mouse chromosome 5. 22 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 22 (Transcript: ENSMUST00000086056). Exon 8~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Piwil1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-437P24 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 targeted null mutation exhibit male sterility due to a block in spermatogenesis beginning at the round spermatid stage.
Exon 8 starts from about 25.41% of the coding region. The knockout of Exon 8~10 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 1112 bp, and the size of intron 10 for 3'-loxP site insertion: 1018 bp. The size of effective cKO region: ~1659 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele 5' gRNA region gRNA region 3'
1 5 6 7 8 9 10 11 12 13 22 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Piwil1 Homology arm cKO region loxP site
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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(8159bp) | A(23.54% 1921) | C(25.44% 2076) | T(24.12% 1968) | G(26.89% 2194)
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.
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BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr5 + 128739978 128742977 3000 browser details YourSeq 128 383 551 3000 92.6% chr18 - 46220710 46220945 236 browser details YourSeq 125 381 551 3000 90.4% chr10 + 71504924 71505120 197 browser details YourSeq 124 387 554 3000 92.0% chr2 + 128897056 128897282 227 browser details YourSeq 124 382 551 3000 91.9% chr19 + 26915398 26915600 203 browser details YourSeq 123 382 551 3000 94.4% chr11 - 43260444 43260614 171 browser details YourSeq 123 382 535 3000 91.9% chr15 + 85970727 85970921 195 browser details YourSeq 122 382 535 3000 94.3% chr12 + 105298466 105298673 208 browser details YourSeq 121 389 551 3000 92.3% chr19 + 36110206 36110412 207 browser details YourSeq 119 382 535 3000 90.5% chr18 - 63399979 63400189 211 browser details YourSeq 119 382 549 3000 91.7% chr7 + 29416636 29416862 227 browser details YourSeq 118 386 535 3000 92.8% chrX + 93710849 93711057 209 browser details YourSeq 117 383 549 3000 92.8% chr1 + 168510450 168510659 210 browser details YourSeq 116 382 551 3000 92.7% chr3 - 86691179 86691402 224 browser details YourSeq 116 383 551 3000 89.1% chr11 - 113593931 113594150 220 browser details YourSeq 116 381 551 3000 93.4% chr11 - 90306615 90306816 202 browser details YourSeq 116 386 535 3000 93.3% chr4 + 58946676 58946854 179 browser details YourSeq 116 386 551 3000 91.0% chr12 + 91508278 91508481 204 browser details YourSeq 115 386 549 3000 91.4% chr2 - 127132888 127133105 218 browser details YourSeq 115 382 563 3000 93.4% chr7 + 131536759 131536975 217
Note: The 3000 bp section upstream of Exon 8 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% chr5 + 128744637 128747636 3000 browser details YourSeq 60 2289 2371 3000 91.7% chr2 + 32830721 32830810 90 browser details YourSeq 59 2279 2371 3000 89.4% chr11 + 85265227 85265350 124 browser details YourSeq 56 2279 2359 3000 91.2% chr9 + 71794878 71794987 110 browser details YourSeq 51 2289 2372 3000 84.8% chr13 - 46947690 46947774 85 browser details YourSeq 51 2289 2373 3000 90.5% chr13 + 77181763 77181851 89 browser details YourSeq 48 2289 2356 3000 90.4% chrY - 2651506 2651571 66 browser details YourSeq 48 2289 2356 3000 90.4% chrY + 2674676 2674741 66 browser details YourSeq 48 2323 2498 3000 90.0% chr15 + 82980881 82981377 497 browser details YourSeq 48 2288 2357 3000 84.3% chr12 + 16788844 16788913 70 browser details YourSeq 47 2289 2357 3000 88.7% chr13 - 104386701 104386767 67 browser details YourSeq 46 2289 2372 3000 83.1% chr18 + 38624028 38624120 93 browser details YourSeq 46 2297 2359 3000 87.1% chr11 + 115907866 115907926 61 browser details YourSeq 45 2287 2357 3000 85.5% chr4 - 100800490 100800558 69 browser details YourSeq 44 2323 2371 3000 95.9% chr11 - 51954138 51954192 55 browser details YourSeq 44 2279 2357 3000 92.2% chr10 - 85248555 85248656 102 browser details YourSeq 42 2291 2355 3000 95.7% chr9 - 45868430 45868816 387 browser details YourSeq 42 2323 2374 3000 92.4% chr14 - 79312940 79313166 227 browser details YourSeq 42 2289 2357 3000 84.0% chr11 + 54995597 54995662 66 browser details YourSeq 41 2289 2341 3000 88.7% chr6 - 72253410 72253462 53
Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Piwil1 piwi-like RNA-mediated gene silencing 1 [ Mus musculus (house mouse) ] Gene ID: 57749, updated on 21-Oct-2019
Gene summary
Official Symbol Piwil1 provided by MGI Official Full Name piwi-like RNA-mediated gene silencing 1 provided by MGI Primary source MGI:MGI:1928897 See related Ensembl:ENSMUSG00000029423 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as MIWI Expression Restricted expression toward testis adult (RPKM 111.5) See more Orthologs human all
Genomic context
Location: 5 G1.3; 5 67.86 cM See Piwil1 in Genome Data Viewer
Exon count: 23
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (128736071..128755474)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (129242121..129261349)
Chromosome 5 - NC_000071.6
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Transcript information: This gene has 3 transcripts
Gene: Piwil1 ENSMUSG00000029423
Description piwi-like RNA-mediated gene silencing 1 [Source:MGI Symbol;Acc:MGI:1928897] Gene Synonyms MIWI Location Chromosome 5: 128,702,524-128,755,474 forward strand. GRCm38:CM000998.2 About this gene This gene has 3 transcripts (splice variants), 213 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Piwil1-201 ENSMUST00000086056.7 4016 862aa ENSMUSP00000083222.3 Protein coding CCDS19690 Q9JMB7 TSL:1 GENCODE basic APPRIS P2
Piwil1-202 ENSMUST00000195959.1 3013 835aa ENSMUSP00000142386.1 Protein coding - Q9JMB7 TSL:1 GENCODE basic APPRIS ALT2
Piwil1-203 ENSMUST00000200192.4 1691 493aa ENSMUSP00000142807.1 Protein coding - A0A0G2JEK6 CDS 3' incomplete TSL:1
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72.95 kb Forward strand 128.70Mb 128.72Mb 128.74Mb 128.76Mb Genes (Comprehensive set... Piwil1-203 >protein coding
Piwil1-201 >protein coding
Piwil1-202 >protein coding
Contigs AC111089.27 > Genes < 4930553I04Rik-201TEC < Rimbp2-205protein coding (Comprehensive set...
< Rimbp2-204protein coding
< Rimbp2-209protein coding
< Rimbp2-201protein coding
< Rimbp2-202protein coding
< Rimbp2-207protein coding
< Rimbp2-208protein coding
Regulatory Build
128.70Mb 128.72Mb 128.74Mb 128.76Mb Reverse strand 72.95 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank
Gene Legend Protein Coding
merged Ensembl/Havana Ensembl protein coding
Non-Protein Coding
processed transcript
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Transcript: ENSMUST00000086056
19.30 kb Forward strand
Piwil1-201 >protein coding
ENSMUSP00000083... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily Ribonuclease H-like superfamily
PAZ domain superfamily SMART GAGE PAZ domain Piwi domain
Pfam GAGE Argonaute, linker 1 domain Piwi domain
PAZ domain PROSITE profiles PAZ domain Piwi domain
PANTHER Piwi-like protein 1
PTHR22891 Gene3D 2.170.260.10 3.40.50.2300 Ribonuclease H superfamily
CDD cd02845 cd04658
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant synonymous variant
Scale bar 0 80 160 240 320 400 480 560 640 720 862
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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