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Mouse Hrk Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Hrk conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Hrk gene (NCBI Reference Sequence: NM_007545 ; Ensembl: ENSMUSG00000046607 ) is located on Mouse chromosome 5. 2 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 1 (Transcript: ENSMUST00000054836). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Hrk gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-313O22 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: Homozygous mutants exhibit motoneurons that are protected from cell death induced by resection of the hypoglossal nerve and delayed cell death of superior cervical ganglia neurons triggered by nerve growth factor withdrawal.
Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~356 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
gRNA region
Wildtype allele gRA NA region T
5' G 3'
1 2
Targeting vector A T G
Targeted allele A T G
Constitutive KO allele (After Cre recombination)
Legends Homology arm Exon of mouse Hrk 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. 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(6276bp) | A(19.76% 1240) | C(29.96% 1880) | T(24.03% 1508) | G(26.26% 1648)
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.
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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 + 118166784 118169783 3000 browser details YourSeq 142 1 189 3000 91.7% chr4 - 72426908 72427474 567 browser details YourSeq 141 1 271 3000 90.9% chr1 - 59587409 59587750 342 browser details YourSeq 141 1 170 3000 92.8% chr17 + 28657427 28657703 277 browser details YourSeq 136 1564 1717 3000 94.2% chr5 + 66177622 66177775 154 browser details YourSeq 136 1 618 3000 81.3% chr19 + 61063369 61063532 164 browser details YourSeq 133 1 628 3000 89.0% chr2 + 119835064 119835683 620 browser details YourSeq 132 1 169 3000 91.3% chr18 + 49627089 49627379 291 browser details YourSeq 132 1 153 3000 93.5% chr1 + 167898196 167898351 156 browser details YourSeq 131 1 150 3000 94.0% chr4 + 59409303 59409473 171 browser details YourSeq 131 1 606 3000 81.0% chr13 + 49771794 49771945 152 browser details YourSeq 129 1 143 3000 95.8% chr2 - 166357086 166357242 157 browser details YourSeq 128 1 139 3000 96.5% chr8 + 119526362 119526504 143 browser details YourSeq 127 13 164 3000 94.5% chr19 - 55246920 55247098 179 browser details YourSeq 126 1 351 3000 87.1% chr6 - 95097309 95097641 333 browser details YourSeq 126 1 141 3000 95.1% chr1 + 33784344 33784488 145 browser details YourSeq 125 7 154 3000 92.6% chr12 - 8927962 8928129 168 browser details YourSeq 125 8 616 3000 80.0% chr15 + 79073219 79073376 158 browser details YourSeq 124 1 313 3000 89.4% chr2 + 30770879 30771189 311 browser details YourSeq 124 5 607 3000 80.7% chr15 + 96467673 96467852 180
Note: The 3000 bp section upstream of Exon 1 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 + 118170060 118173059 3000 browser details YourSeq 198 225 565 3000 92.3% chr4 - 101312771 101313157 387 browser details YourSeq 165 251 531 3000 88.4% chr4 + 126274726 126275036 311 browser details YourSeq 163 227 899 3000 80.2% chr12 + 17180242 17180486 245 browser details YourSeq 159 249 565 3000 90.7% chr3 + 100305402 100305850 449 browser details YourSeq 148 348 899 3000 80.6% chr15 + 68499846 68500101 256 browser details YourSeq 123 223 591 3000 81.7% chr10 - 41604230 41604511 282 browser details YourSeq 106 344 635 3000 77.0% chr6 + 32111809 32111973 165 browser details YourSeq 103 146 354 3000 87.5% chrX - 103651962 103652163 202 browser details YourSeq 103 259 506 3000 80.0% chr13 - 47585408 47585557 150 browser details YourSeq 102 345 856 3000 77.1% chr9 - 98156521 98156694 174 browser details YourSeq 99 219 560 3000 81.4% chr2 + 97489139 97489350 212 browser details YourSeq 97 454 879 3000 76.1% chr1 + 68947732 68947953 222 browser details YourSeq 95 219 891 3000 74.6% chr2 - 71911262 71911381 120 browser details YourSeq 95 226 354 3000 95.4% chr13 + 51279122 51279318 197 browser details YourSeq 95 253 397 3000 92.3% chr1 + 175217958 175218109 152 browser details YourSeq 95 355 654 3000 78.6% chr1 + 23795859 23795993 135 browser details YourSeq 93 347 848 3000 76.9% chr1 + 158396090 158396247 158 browser details YourSeq 91 219 799 3000 75.6% chr1 - 15918413 15918513 101 browser details YourSeq 90 219 443 3000 82.0% chr1 - 23104410 23104518 109
Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Hrk harakiri, BCL2 interacting protein (contains only BH3 domain) [ Mus musculus (house mouse) ] Gene ID: 12123, updated on 12-Aug-2019
Gene summary
Official Symbol Hrk provided by MGI Official Full Name harakiri, BCL2 interacting protein (contains only BH3 domain) provided by MGI Primary source MGI:MGI:1201608 See related Ensembl:ENSMUSG00000046607 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 DP5; Bid3; AI838259; harakiri Expression Biased expression in frontal lobe adult (RPKM 5.4), cortex adult (RPKM 4.2) and 6 other tissues See more Orthologs human all
Genomic context
Location: 5; 5 F See Hrk in Genome Data Viewer
Exon count: 2
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (118169764..118189478)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (118619773..118639487)
Chromosome 5 - NC_000071.6
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Transcript information: This gene has 2 transcripts
Gene: Hrk ENSMUSG00000046607
Description harakiri, BCL2 interacting protein (contains only BH3 domain) [Source:MGI Symbol;Acc:MGI:1201608] Gene Synonyms Bid3, DP5 Location Chromosome 5: 118,164,648-118,189,478 forward strand. GRCm38:CM000998.2 About this gene This gene has 2 transcripts (splice variants), 46 orthologues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Hrk-201 ENSMUST00000054836.6 5382 92aa ENSMUSP00000057532.4 Protein coding CCDS39236 P62816 TSL:1 GENCODE basic APPRIS P1
Hrk-202 ENSMUST00000184679.1 646 No protein - lncRNA - - TSL:3
44.83 kb Forward strand 118.16Mb 118.17Mb 118.18Mb 118.19Mb Genes (Comprehensive set... Hrk-202 >lncRNA
Hrk-201 >protein coding
Contigs < AC110254.11 Genes < Fbxw8-201protein coding < Rnft2-201protein coding (Comprehensive set...
< Fbxw8-202retained intron < Rnft2-202protein coding
< Rnft2-205retained intron
< Rnft2-203protein coding
Regulatory Build
118.16Mb 118.17Mb 118.18Mb 118.19Mb Reverse strand 44.83 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
processed transcript RNA gene
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Transcript: ENSMUST00000054836
19.73 kb Forward strand
Hrk-201 >protein coding
ENSMUSP00000057... Low complexity (Seg) Pfam Activator of apoptosis harakiri PROSITE patterns Apoptosis regulator, Bcl-2, BH3 motif, conserved site PIRSF Activator of apoptosis harakiri PANTHER Activator of apoptosis harakiri
All sequence SNPs/i... Sequence variants (dbSNP and all other sources) R S R Y
Variant Legend missense variant synonymous variant
Scale bar 0 8 16 24 32 40 48 56 64 72 80 92
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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