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Mouse Klc2 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Klc2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Klc2 gene (NCBI Reference Sequence: NM_008451 ; Ensembl: ENSMUSG00000024862 ) is located on Mouse chromosome 19. 16 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 16 (Transcript: ENSMUST00000116563). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Klc2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-41O10 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 3 starts from about 12.33% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 3022 bp, and the size of intron 4 for 3'-loxP site insertion: 840 bp. The size of effective cKO region: ~891 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 3 4 5 6 7 8 16 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Klc2 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(7391bp) | A(26.1% 1929) | C(24.14% 1784) | T(22.62% 1672) | G(27.14% 2006)
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% chr19 - 5114422 5117421 3000 browser details YourSeq 247 1808 2465 3000 94.0% chr9 + 88343882 88561327 217446 browser details YourSeq 232 1585 2291 3000 87.9% chr2 - 93417723 93579042 161320 browser details YourSeq 201 1509 2059 3000 94.4% chr1 - 74309418 74675151 365734 browser details YourSeq 168 2189 2487 3000 87.9% chr3 + 9166874 9365125 198252 browser details YourSeq 148 1613 2066 3000 82.3% chr19 - 4031009 4031331 323 browser details YourSeq 142 1948 2258 3000 88.4% chr10 + 42451544 42588261 136718 browser details YourSeq 138 2191 2426 3000 87.9% chr10 + 61602037 61602246 210 browser details YourSeq 134 1656 2378 3000 91.0% chr8 - 18585786 18586871 1086 browser details YourSeq 132 1553 1846 3000 82.8% chr4 - 133138691 133138906 216 browser details YourSeq 131 2311 2463 3000 95.3% chr7 - 34541638 34541801 164 browser details YourSeq 131 1488 2066 3000 91.8% chr1 + 132943424 133197882 254459 browser details YourSeq 130 1671 1802 3000 99.3% chr19 - 5114376 5114507 132 browser details YourSeq 127 2331 2490 3000 91.3% chr13 - 15205583 15205741 159 browser details YourSeq 125 2324 2465 3000 95.0% chr11 - 79813551 79813702 152 browser details YourSeq 125 2031 2462 3000 83.3% chr1 - 71635416 71635784 369 browser details YourSeq 121 2326 2461 3000 94.9% chr2 - 92653118 92653262 145 browser details YourSeq 118 2306 2465 3000 89.1% chr14 + 91979431 91979589 159 browser details YourSeq 118 2334 2465 3000 95.5% chr13 + 54116349 54116491 143 browser details YourSeq 117 2325 2477 3000 95.5% chr2 - 18796402 18796599 198
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% chr19 - 5110531 5113530 3000 browser details YourSeq 266 635 2066 3000 84.9% chr17 - 46636720 46640069 3350 browser details YourSeq 239 974 2352 3000 87.2% chr12 + 111553526 111782002 228477 browser details YourSeq 238 632 2453 3000 80.4% chr7 - 19395384 19397053 1670 browser details YourSeq 139 251 402 3000 96.1% chr17 + 86647830 86647982 153 browser details YourSeq 133 251 396 3000 95.9% chr2 - 32729220 32729371 152 browser details YourSeq 133 251 397 3000 95.3% chr19 - 5076112 5076258 147 browser details YourSeq 133 251 402 3000 91.2% chr10 - 128055476 128055622 147 browser details YourSeq 132 251 396 3000 93.8% chr5 + 107472350 107472494 145 browser details YourSeq 131 252 402 3000 94.1% chr15 - 24902285 24902437 153 browser details YourSeq 131 251 389 3000 97.2% chr7 + 114107461 114107599 139 browser details YourSeq 128 259 396 3000 96.4% chr8 - 60538402 60538539 138 browser details YourSeq 128 252 397 3000 94.6% chr18 - 26445444 26445591 148 browser details YourSeq 127 252 396 3000 93.8% chr19 - 10874758 10874902 145 browser details YourSeq 127 960 1109 3000 92.5% chr6 + 120561594 120561742 149 browser details YourSeq 126 257 396 3000 95.0% chr4 - 18230078 18230217 140 browser details YourSeq 126 259 396 3000 96.4% chr12 + 17427206 17427347 142 browser details YourSeq 125 255 397 3000 91.5% chr11 + 77074582 77074721 140 browser details YourSeq 123 253 391 3000 94.3% chr1 - 87073439 87073577 139 browser details YourSeq 123 256 389 3000 94.0% chr1 - 71766596 71766727 132
Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Klc2 kinesin light chain 2 [ Mus musculus (house mouse) ] Gene ID: 16594, updated on 24-Oct-2019
Gene summary
Official Symbol Klc2 provided by MGI Official Full Name kinesin light chain 2 provided by MGI Primary source MGI:MGI:107953 See related Ensembl:ENSMUSG00000024862 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 KLC 2; KLC-2; AW212649; 8030455F02Rik Expression Broad expression in cerebellum adult (RPKM 59.4), cortex adult (RPKM 42.0) and 24 other tissues See more Orthologs human all
Genomic context
Location: 19 A; 19 4.25 cM See Klc2 in Genome Data Viewer
Exon count: 16
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (5107746..5118298, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (5107746..5118408, complement)
Chromosome 19 - NC_000085.6
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Transcript information: This gene has 9 transcripts
Gene: Klc2 ENSMUSG00000024862
Description kinesin light chain 2 [Source:MGI Symbol;Acc:MGI:107953] Gene Synonyms 8030455F02Rik Location Chromosome 19: 5,107,746-5,118,560 reverse strand. GRCm38:CM001012.2 About this gene This gene has 9 transcripts (splice variants), 161 orthologues, 6 paralogues, 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
Klc2-204 ENSMUST00000116563.7 3169 619aa ENSMUSP00000112262.1 Protein coding CCDS37889 Q91YS4 TSL:1 GENCODE basic APPRIS P1
Klc2-201 ENSMUST00000025798.12 2940 617aa ENSMUSP00000025798.6 Protein coding - D3YXZ3 TSL:5 GENCODE basic
Klc2-202 ENSMUST00000113727.7 2874 617aa ENSMUSP00000109356.1 Protein coding - D3YXZ3 TSL:5 GENCODE basic
Klc2-203 ENSMUST00000113728.7 2838 617aa ENSMUSP00000109357.1 Protein coding - D3YXZ3 TSL:5 GENCODE basic
Klc2-209 ENSMUST00000156717.1 665 166aa ENSMUSP00000122458.1 Protein coding - D3Z5Y7 CDS 3' incomplete TSL:5
Klc2-206 ENSMUST00000137403.7 841 No protein - Retained intron - - TSL:2
Klc2-208 ENSMUST00000149806.1 770 No protein - Retained intron - - TSL:2
Klc2-207 ENSMUST00000142255.1 758 No protein - Retained intron - - TSL:2
Klc2-205 ENSMUST00000135827.1 411 No protein - Retained intron - - TSL:5
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30.82 kb Forward strand
5.10Mb 5.11Mb 5.12Mb Genes Gm10817-201 >lncRNA (Comprehensive set...
Contigs < AC124502.4 < AC125059.4 Genes (Comprehensive set... < Cnih2-201protein coding < Klc2-204protein coding
< Cnih2-202nonsense mediated decay < Klc2-201protein coding
< Rab1b-201protein coding < Klc2-203protein coding
< Rab1b-204protein coding < Klc2-202protein coding
< Rab1b-202protein coding < Klc2-207retained intron < Klc2-205retained intron
< Rab1b-203protein coding < Klc2-206retained intron < Klc2-209protein coding
< Klc2-208retained intron
Regulatory Build
5.10Mb 5.11Mb 5.12Mb Reverse strand 30.82 kb
Regulation Legend CTCF Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
merged Ensembl/Havana Ensembl protein coding
Non-Protein Coding
processed transcript RNA gene
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Transcript: ENSMUST00000116563
< Klc2-204protein coding
Reverse strand 10.81 kb
ENSMUSP00000112... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat Prints Kinesin light chain Pfam PF13424 PF13374 PROSITE profiles Tetratricopeptide repeat
Tetratricopeptide repeat-containing domain PROSITE patterns Kinesin light chain repeat PANTHER PTHR45783:SF2
PTHR45783 Gene3D Tetratricopeptide-like helical domain superfamily
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend inframe insertion missense variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 480 540 619
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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