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Mouse Cct4 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Cct4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Cct4 gene (NCBI Reference Sequence: NM_009837 ; Ensembl: ENSMUSG00000007739 ) is located on Mouse chromosome 11. 14 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 14 (Transcript: ENSMUST00000173867). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cct4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-214C7 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 2 starts from about 7.92% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2386 bp, and the size of intron 3 for 3'-loxP site insertion: 1542 bp. The size of effective cKO region: ~1621 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 2 3 4 5 14 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Cct4 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(8121bp) | A(28.83% 2341) | C(19.15% 1555) | T(30.23% 2455) | G(21.8% 1770)
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% chr11 + 22990018 22993017 3000 browser details YourSeq 109 621 853 3000 78.9% chr7 - 25443454 25443600 147 browser details YourSeq 79 2038 2155 3000 87.9% chr1 + 118408497 118738433 329937 browser details YourSeq 76 1954 2082 3000 83.7% chr7 - 127526490 127526616 127 browser details YourSeq 76 1993 2107 3000 91.5% chr12 - 91783131 91783247 117 browser details YourSeq 76 1955 2107 3000 94.3% chr2 + 29824786 29824938 153 browser details YourSeq 74 2031 2169 3000 93.2% chrX + 60422114 60422451 338 browser details YourSeq 74 2123 2237 3000 82.7% chr5 + 131412696 131412811 116 browser details YourSeq 72 1951 2079 3000 94.0% chr8 - 125879570 125879700 131 browser details YourSeq 72 1949 2082 3000 92.0% chr8 - 83734665 83734798 134 browser details YourSeq 72 1998 2149 3000 91.0% chr3 + 31577844 31578090 247 browser details YourSeq 71 1993 2118 3000 91.9% chr8 - 69745939 69746066 128 browser details YourSeq 69 1998 2117 3000 89.1% chr10 + 63119621 63119739 119 browser details YourSeq 67 1948 2075 3000 90.6% chr8 - 22494075 22494204 130 browser details YourSeq 67 2038 2324 3000 94.9% chr11 - 65091377 65091793 417 browser details YourSeq 67 1954 2079 3000 93.6% chr4 + 34626666 34626793 128 browser details YourSeq 66 2123 2329 3000 92.5% chrX + 99366565 99366774 210 browser details YourSeq 65 1955 2079 3000 92.4% chr5 - 125439671 125439797 127 browser details YourSeq 65 1993 2118 3000 85.4% chr18 - 66418270 66418393 124 browser details YourSeq 65 2118 2237 3000 77.5% chr1 - 69830511 69830631 121
Note: The 3000 bp section upstream of Exon 2 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 + 22994639 22997638 3000 browser details YourSeq 300 1294 2513 3000 85.5% chr7 - 25443070 25443434 365 browser details YourSeq 244 421 1062 3000 93.0% chr1 - 58350035 58837847 487813 browser details YourSeq 209 409 1064 3000 91.7% chr2 + 33317596 33469222 151627 browser details YourSeq 203 450 1067 3000 82.6% chr7 + 142850392 142850812 421 browser details YourSeq 195 426 1064 3000 89.4% chr8 + 110987406 111109706 122301 browser details YourSeq 189 407 1032 3000 92.4% chr1 + 84870471 84926139 55669 browser details YourSeq 177 407 994 3000 88.4% chr13 - 62638356 62638937 582 browser details YourSeq 176 445 1063 3000 82.8% chr2 - 69872014 69872372 359 browser details YourSeq 175 432 1010 3000 94.5% chr1 + 20994699 21404096 409398 browser details YourSeq 138 447 1031 3000 82.1% chr9 - 86516284 86516764 481 browser details YourSeq 138 389 559 3000 94.9% chr16 + 65478609 65478795 187 browser details YourSeq 138 406 590 3000 92.6% chr1 + 68677761 68677955 195 browser details YourSeq 136 406 558 3000 94.8% chr16 - 30497807 30497960 154 browser details YourSeq 135 393 560 3000 90.2% chr4 - 133664073 133664234 162 browser details YourSeq 132 407 560 3000 90.7% chr1 - 52882608 52882756 149 browser details YourSeq 131 407 559 3000 90.6% chr1 - 72818255 72818402 148 browser details YourSeq 130 407 559 3000 90.0% chr3 + 113161739 113161887 149 browser details YourSeq 129 407 557 3000 93.4% chr14 - 40970005 40970157 153 browser details YourSeq 129 407 559 3000 92.2% chr11 - 114937019 114937171 153
Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Cct4 chaperonin containing Tcp1, subunit 4 (delta) [ Mus musculus (house mouse) ] Gene ID: 12464, updated on 12-Aug-2019
Gene summary
Official Symbol Cct4 provided by MGI Official Full Name chaperonin containing Tcp1, subunit 4 (delta) provided by MGI Primary source MGI:MGI:104689 See related Ensembl:ENSMUSG00000007739 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 A45; Cctd; C78323; 2610204B21Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 82.4), placenta adult (RPKM 69.7) and 28 other tissues See more Orthologs human all
Genomic context
Location: 11 A3.2; 11 14.25 cM See Cct4 in Genome Data Viewer
Exon count: 14
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (22990593..23003336)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (22890593..22903336)
Chromosome 11 - NC_000077.6
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Transcript information: This gene has 9 transcripts
Gene: Cct4 ENSMUSG00000007739
Description chaperonin containing Tcp1, subunit 4 (delta) [Source:MGI Symbol;Acc:MGI:104689] Gene Synonyms 2610204B21Rik, A45, Cctd, T complex protein 1, delta, TCP-1 delta Location Chromosome 11: 22,990,519-23,003,780 forward strand. GRCm38:CM001004.2 About this gene This gene has 9 transcripts (splice variants), 212 orthologues, 13 paralogues and is a member of 1 Ensembl protein family. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Cct4- ENSMUST00000173867.7 2484 539aa ENSMUSP00000133523.1 Protein coding CCDS24474 P80315 TSL:1 204 Q564F4 GENCODE basic APPRIS P1
Cct4- ENSMUST00000020562.4 1634 509aa ENSMUSP00000020562.4 Protein coding - G5E839 TSL:5 201 GENCODE basic
Cct4- ENSMUST00000174047.7 581 120aa ENSMUSP00000134248.1 Nonsense mediated - G3UYW5 TSL:5 205 decay
Cct4- ENSMUST00000174659.1 489 47aa ENSMUSP00000133667.1 Nonsense mediated - G3UXF3 TSL:5 207 decay
Cct4- ENSMUST00000173853.7 465 59aa ENSMUSP00000133677.1 Nonsense mediated - G3UXG2 TSL:3 203 decay
Cct4- ENSMUST00000174244.7 1736 No - Retained intron - - TSL:1 206 protein
Cct4- ENSMUST00000174746.1 627 No - Retained intron - - TSL:1 209 protein
Cct4- ENSMUST00000174689.1 507 No - Retained intron - - TSL:2 208 protein
Cct4- ENSMUST00000145912.1 596 No - lncRNA - - TSL:2 202 protein
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33.26 kb Forward strand 22.99Mb 23.00Mb 23.01Mb Genes (Comprehensive set... Cct4-204 >protein coding Fam161a-206 >protein coding
Cct4-203 >nonsense mediated decay Cct4-202 >lncRNA Fam161a-205 >protein coding
Cct4-206 >retained intron Cct4-208 >retained intron Fam161a-203 >protein coding
Cct4-201 >protein coding Fam161a-208 >protein coding
Cct4-205 >nonsense mediated decay Fam161a-201 >protein coding
Cct4-207 >nonsense mediated decay
Cct4-209 >retained intron
Contigs BX001008.3 >
Genes < Gm28048-201protein coding (Comprehensive set...
< Commd1-204protein coding
< Commd1-202protein coding
< Commd1-203protein coding
Regulatory Build
22.99Mb 23.00Mb 23.01Mb Reverse strand 33.26 kb
Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site
Gene Legend Protein Coding
merged Ensembl/Havana Ensembl protein coding
Non-Protein Coding
processed transcript RNA gene
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Transcript: ENSMUST00000173867
13.26 kb Forward strand
Cct4-204 >protein coding
ENSMUSP00000133... MobiDB lite TIGRFAM T-complex protein 1, delta subunit Superfamily GroEL-like apical domain superfamily
GroEL-like equatorial domain superfamily
TCP-1-like chaperonin intermediate domain superfamily Prints Chaperone tailless complex polypeptide 1 (TCP-1) Pfam Chaperonin Cpn60/TCP-1 family
PROSITE patterns Chaperonin TCP-1, conserved site
Chaperonin TCP-1, conserved site
Chaperonin TCP-1, conserved site PANTHER PTHR11353:SF26
PTHR11353 Gene3D GroEL-like equatorial domain superfamily
GroEL-like apical domain superfamily CDD T-complex protein 1, delta subunit
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 539
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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