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Mouse Cct3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cct3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cct3 (NCBI Reference Sequence: NM_009836 ; Ensembl: ENSMUSG00000001416 ) is located on Mouse 3. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000001452). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cct3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-96H9 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: Mice homozygous for a transgenic gene disruption may exhibit lethality at E8.

Exon 5 starts from about 12.72% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1776 bp, and the size of intron 6 for 3'-loxP site insertion: 4030 bp. The size of effective cKO region: ~2863 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 3 4 5 6 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cct3 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(9363bp) | A(27.15% 2542) | C(20.77% 1945) | T(30.45% 2851) | G(21.63% 2025)

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% chr3 + 88299519 88302518 3000 browser details YourSeq 337 601 2561 3000 93.6% chr7 + 15894107 16042055 147949 browser details YourSeq 229 1280 2573 3000 94.6% chr11 - 90624902 90861314 236413 browser details YourSeq 185 594 889 3000 91.0% chr9 - 113993159 113993510 352 browser details YourSeq 141 2427 2587 3000 95.6% chr10 - 82332008 82332182 175 browser details YourSeq 139 2426 2573 3000 97.3% chr18 + 67452481 67452630 150 browser details YourSeq 138 2427 2573 3000 97.3% chr10 - 7811988 7812142 155 browser details YourSeq 138 596 756 3000 93.2% chr15 + 85775870 85776034 165 browser details YourSeq 138 798 1065 3000 77.2% chr1 + 36008156 36246320 238165 browser details YourSeq 137 591 888 3000 83.5% chr18 - 67791207 67791449 243 browser details YourSeq 136 2427 2573 3000 96.6% chr11 - 113524488 113524636 149 browser details YourSeq 136 2427 2571 3000 97.3% chr11 - 78654395 78654546 152 browser details YourSeq 136 2427 2574 3000 96.0% chr1 - 72315269 72315416 148 browser details YourSeq 136 625 837 3000 90.0% chr19 + 37212159 37212796 638 browser details YourSeq 136 2426 2573 3000 96.7% chr11 + 82258649 82258805 157 browser details YourSeq 136 594 758 3000 91.4% chr10 + 112807608 112807773 166 browser details YourSeq 135 2427 2574 3000 96.0% chr8 - 109786986 109787146 161 browser details YourSeq 135 2427 2573 3000 96.0% chr16 - 35072255 35072401 147 browser details YourSeq 135 594 759 3000 89.5% chr11 - 101346642 101346805 164 browser details YourSeq 135 2430 2573 3000 97.3% chr15 + 59101634 59101784 151

Note: The 3000 bp section upstream of Exon 5 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% chr3 + 88305382 88308381 3000 browser details YourSeq 298 1870 2239 3000 93.6% chr3 + 94617232 94617600 369 browser details YourSeq 287 1877 2227 3000 92.2% chr11 + 5719778 5720136 359 browser details YourSeq 286 1868 2210 3000 94.5% chr11 - 53712046 53712399 354 browser details YourSeq 285 1877 2219 3000 93.4% chr9 - 121307688 121308047 360 browser details YourSeq 284 1870 2217 3000 94.1% chr12 - 80496271 80496655 385 browser details YourSeq 281 1869 2211 3000 93.6% chr17 + 78776554 78776898 345 browser details YourSeq 271 1879 2211 3000 93.6% chr17 - 27532560 27532954 395 browser details YourSeq 269 1869 2210 3000 93.3% chr10 - 77148651 77149032 382 browser details YourSeq 267 1874 2237 3000 93.0% chrX + 52757481 52757922 442 browser details YourSeq 263 1878 2210 3000 92.6% chr7 + 44883670 44884017 348 browser details YourSeq 261 1878 2210 3000 94.3% chr11 - 120735961 120736334 374 browser details YourSeq 245 2017 2611 3000 90.5% chr17 + 66319141 66319661 521 browser details YourSeq 240 2017 2611 3000 87.3% chr9 + 82092652 82093028 377 browser details YourSeq 238 1869 2209 3000 93.5% chr2 + 120000196 120000561 366 browser details YourSeq 235 2017 2602 3000 88.9% chr9 + 10109468 10109860 393 browser details YourSeq 229 1924 2208 3000 96.4% chr9 - 72794265 72794679 415 browser details YourSeq 221 2027 2611 3000 87.5% chr3 - 76678811 76679171 361 browser details YourSeq 219 1958 2239 3000 96.2% chr16 + 36028090 36028595 506 browser details YourSeq 213 2030 2600 3000 85.7% chr19 - 50779625 50779892 268

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Cct3 containing Tcp1, subunit 3 (gamma) [ Mus musculus (house mouse) ] Gene ID: 12462, updated on 12-Aug-2019

Gene summary

Official Symbol Cct3 provided by MGI Official Full Name chaperonin containing Tcp1, subunit 3 (gamma) provided by MGI Primary source MGI:MGI:104708 See related Ensembl:ENSMUSG00000001416 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 Cctg; TriC-P5; AL024092; Tcp1-rs3 Expression Ubiquitous expression in CNS E11.5 (RPKM 119.9), placenta adult (RPKM 98.5) and 28 other tissues See more Orthologs human all

Genomic context

Location: 3 F1; 3 38.79 cM See Cct3 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (88297135..88321766)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (88101057..88125688)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 10 transcripts

Gene: Cct3 ENSMUSG00000001416

Description chaperonin containing Tcp1, subunit 3 (gamma) [Source:MGI Symbol;Acc:MGI:104708] Gene Synonyms Cctg, Tcp1-rs3, TriC-P5 Location Chromosome 3: 88,297,116-88,321,767 forward strand. GRCm38:CM000996.2 About this gene This gene has 10 transcripts (splice variants), 222 orthologues, 13 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cct3- ENSMUST00000001452.13 1977 545aa ENSMUSP00000001452.7 Protein CCDS17468 P80318 TSL:1 201 coding Q3U4U6 GENCODE basic APPRIS P1

Cct3- ENSMUST00000168062.7 1943 521aa ENSMUSP00000131113.1 Protein - Q3U0I3 TSL:1 208 coding GENCODE basic

Cct3- ENSMUST00000164166.7 1804 507aa ENSMUSP00000126109.1 Protein - E9Q133 TSL:5 204 coding GENCODE basic

Cct3- ENSMUST00000163735.1 919 306aa ENSMUSP00000130616.1 Protein - F6Q609 CDS 5' and 3' 202 coding incomplete TSL:3

Cct3- ENSMUST00000168971.1 474 85aa ENSMUSP00000131250.1 Protein - F6ZVG8 CDS 5' incomplete 209 coding TSL:3

Cct3- ENSMUST00000167000.7 3335 No - Retained - - TSL:5 206 protein intron

Cct3- ENSMUST00000167718.1 2056 No - Retained - - TSL:2 207 protein intron

Cct3- ENSMUST00000164783.7 1932 No - Retained - - TSL:2 205 protein intron

Cct3- ENSMUST00000193666.1 1831 No - Retained - - TSL:NA 210 protein intron

Cct3- ENSMUST00000164122.1 472 No - Retained - - TSL:2 203 protein intron

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44.65 kb Forward strand

88.29Mb 88.30Mb 88.31Mb 88.32Mb 88.33Mb (Comprehensive set... Cct3-201 >protein coding Glmp-202 >nonsense mediated decay

Cct3-210 >retained intron Cct3-203 >retained intron

Cct3-206 >retained intron Glmp-209 >protein coding

Cct3-205 >retained intron Cct3-209 >protein coding

Cct3-207 >retained intron Glmp-210 >retained intron

Cct3-204 >protein coding Glmp-205 >retained intron

Cct3-208 >protein coding Glmp-204 >retained intron

Cct3-202 >protein coding Glmp-207 >protein coding

Glmp-201 >protein coding

Glmp-208 >protein coding

Glmp-203 >retained intron

Glmp-206 >protein coding

Contigs AC044864.6 > Genes < Tsacc-202protein coding < Tmem79-201protein coding (Comprehensive set...

< Tsacc-201protein coding < Tmem79-203protein coding

< Gm38392-202protein coding < Tsacc-203retained intron < Tmem79-202protein coding

< Gm38392-201nonsense mediated decay

Regulatory Build

88.29Mb 88.30Mb 88.31Mb 88.32Mb 88.33Mb Reverse strand 44.65 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000001452

24.65 kb Forward strand

Cct3-201 >protein coding

ENSMUSP00000001... PDB-ENSP mappings MobiDB lite TIGRFAM T-complex protein 1, gamma subunit Superfamily GroEL-like equatorial domain superfamily

GroEL-like apical 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

PTHR11353:SF24 Gene3D TCP-1-like chaperonin intermediate domain superfamily

GroEL-like apical domain superfamily

GroEL-like equatorial domain superfamily CDD T-complex protein 1, gamma subunit

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 545

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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