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Mouse Tbcb Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Tbcb gene (NCBI Reference Sequence: NM_025548 ; Ensembl: ENSMUSG00000006095 ) is located on Mouse chromosome 7. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000006254). 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 Tbcb gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-119L7 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 35.38% 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: 3425 bp, and the size of intron 4 for 3'-loxP site insertion: 2298 bp. The size of effective cKO region: ~1357 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 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tbcb 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. 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(7857bp) | A(25.43% 1998) | C(22.4% 1760) | T(26.83% 2108) | G(25.34% 1991)

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% chr7 - 30227951 30230950 3000 browser details YourSeq 288 2020 2389 3000 94.3% chr8 - 123973946 123974309 364 browser details YourSeq 287 2034 2393 3000 95.6% chr8 - 4598993 4599508 516 browser details YourSeq 279 2034 2389 3000 92.8% chr15 + 8125943 8126296 354 browser details YourSeq 278 2026 2401 3000 90.0% chr15 - 55551835 55552212 378 browser details YourSeq 272 2029 2396 3000 90.6% chr9 - 64248855 64249208 354 browser details YourSeq 270 2029 2389 3000 94.5% chr17 + 56301390 56646974 345585 browser details YourSeq 264 2029 2389 3000 93.0% chr11 - 69816173 69816505 333 browser details YourSeq 263 2030 2389 3000 94.0% chr1 + 136755567 136755938 372 browser details YourSeq 258 2029 2390 3000 97.1% chr10 + 128273375 128717615 444241 browser details YourSeq 256 2029 2389 3000 93.6% chr15 + 81552577 81552945 369 browser details YourSeq 255 2026 2382 3000 90.1% chr1 + 22129434 22129754 321 browser details YourSeq 254 2043 2389 3000 94.8% chr2 - 30855247 30855644 398 browser details YourSeq 247 2054 2389 3000 94.2% chr10 - 61397071 61397405 335 browser details YourSeq 246 2053 2389 3000 94.3% chr17 + 46817001 46817339 339 browser details YourSeq 244 2030 2390 3000 93.3% chr12 + 13001405 13001756 352 browser details YourSeq 242 2049 2389 3000 93.6% chr1 + 179682310 179682901 592 browser details YourSeq 235 1862 2389 3000 91.1% chr9 - 120606606 120607095 490 browser details YourSeq 228 2075 2389 3000 92.9% chr7 - 45104764 45105436 673 browser details YourSeq 228 2074 2389 3000 92.9% chr5 + 120638526 120639196 671

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% chr7 - 30223594 30226593 3000 browser details YourSeq 396 721 1225 3000 90.8% chr7 + 44566056 44963511 397456 browser details YourSeq 347 1038 1661 3000 93.5% chr6 - 30055759 30124226 68468 browser details YourSeq 327 1001 1398 3000 93.4% chr11 + 97945510 97945913 404 browser details YourSeq 309 1019 1400 3000 92.2% chr2 + 158293794 158294201 408 browser details YourSeq 304 1020 1393 3000 94.8% chr2 + 92134953 92135335 383 browser details YourSeq 300 1020 1382 3000 93.4% chr7 - 45478137 45478505 369 browser details YourSeq 297 1039 1717 3000 90.1% chr17 + 71484087 71484597 511 browser details YourSeq 293 1039 1703 3000 90.1% chr17 + 53548466 53549047 582 browser details YourSeq 292 1039 1703 3000 89.6% chr5 + 23823657 23824003 347 browser details YourSeq 288 1039 1717 3000 88.0% chr8 - 70619962 70620335 374 browser details YourSeq 284 1039 1703 3000 87.7% chr11 + 6609582 6609966 385 browser details YourSeq 283 1039 1717 3000 87.0% chr18 - 77874992 77875350 359 browser details YourSeq 276 1039 1398 3000 92.6% chrX + 105961512 105962124 613 browser details YourSeq 275 1039 1697 3000 89.0% chr10 - 128222946 128223505 560 browser details YourSeq 275 1037 1716 3000 85.4% chr12 + 111513281 111513634 354 browser details YourSeq 265 1039 1698 3000 88.6% chr11 + 78312121 78312629 509 browser details YourSeq 264 1038 1393 3000 93.5% chr3 + 86893086 86893639 554 browser details YourSeq 264 1000 1701 3000 88.4% chr11 + 89045491 89046100 610 browser details YourSeq 262 1038 1393 3000 93.2% chr4 - 155497325 155497921 597

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: Tbcb tubulin folding cofactor B [ Mus musculus (house mouse) ] Gene ID: 66411, updated on 12-Aug-2019

Gene summary

Official Symbol Tbcb provided by MGI Official Full Name tubulin folding cofactor B provided by MGI Primary source MGI:MGI:1913661 See related Ensembl:ENSMUSG00000006095 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 CG22; CKAPI; Ckap1; AU041393; 2410007D12Rik Expression Ubiquitous expression in CNS E18 (RPKM 59.9), CNS E14 (RPKM 49.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 B1 See Tbcb in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (30224129..30232197, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (31009150..31017048, complement)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 3 transcripts

Gene: Tbcb ENSMUSG00000006095

Description tubulin folding cofactor B [Source:MGI Symbol;Acc:MGI:1913661] Gene Synonyms 2410007D12Rik, Ckap1 Location Chromosome 7: 30,224,131-30,232,272 reverse strand. GRCm38:CM001000.2 About this gene This gene has 3 transcripts (splice variants), 183 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tbcb-201 ENSMUST00000006254.5 1416 244aa ENSMUSP00000006254.5 Protein coding CCDS21083 Q9D1E6 TSL:1 GENCODE basic APPRIS P1

Tbcb-202 ENSMUST00000141608.1 502 No protein - Retained intron - - TSL:1

Tbcb-203 ENSMUST00000169358.7 396 No protein - lncRNA - - TSL:5

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28.14 kb Forward strand 30.22Mb 30.23Mb 30.24Mb Genes Polr2i-201 >protein coding (Comprehensive set...

Polr2i-207 >protein coding

Polr2i-202 >protein coding

Polr2i-203 >protein coding

Polr2i-206 >retained intron

Polr2i-204 >retained intron

Polr2i-208 >lncRNA

Polr2i-205 >retained intron

Contigs AC149067.3 > Genes (Comprehensive set... < Tbcb-201protein coding < Ovol3-201protein coding < Wdr62-207retained intron

< Tbcb-203lncRNA < Wdr62-205nonsense mediated decay

< Tbcb-202retained intron < Wdr62-203retained intron

< Wdr62-201protein coding

< Wdr62-204protein coding

Regulatory Build

30.22Mb 30.23Mb 30.24Mb Reverse strand 28.14 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: ENSMUST00000006254

< Tbcb-201protein coding

Reverse strand 8.14 kb

ENSMUSP00000006... PDB-ENSP mappings Low complexity (Seg) Superfamily Ubiquitin-like domain superfamily CAP Gly-rich domain superfamily

SMART CAP Gly-rich domain Pfam Ubiquitin domain CAP Gly-rich domain

PROSITE profiles CAP Gly-rich domain PROSITE patterns CAP Gly-rich domain PANTHER PTHR18916:SF6

PTHR18916 Gene3D 3.10.20.90 CAP Gly-rich domain superfamily

CDD cd01789

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 244

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

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