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Mouse Tbrg4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tbrg4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tbrg4 gene (NCBI Reference Sequence: NM_001130457 ; Ensembl: ENSMUSG00000000384 ) is located on Mouse chromosome 11. 13 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000189268). Exon 7~12 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tbrg4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-69P8 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 7 starts from about 56.08% of the coding region. The knockout of Exon 7~12 will result in frameshift of the gene. The size of intron 6 for 5'-loxP site insertion: 445 bp, and the size of intron 12 for 3'-loxP site insertion: 791 bp. The size of effective cKO region: ~2967 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 4 5 6 7 8 9 10 11 12 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tbrg4 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(9439bp) | A(22.48% 2122) | C(25.02% 2362) | T(26.77% 2527) | G(25.72% 2428)

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% chr11 - 6619333 6622332 3000 browser details YourSeq 136 1877 2016 3000 98.6% chr6 + 70855442 70855581 140 browser details YourSeq 109 1879 2102 3000 80.6% chr3 - 96724197 96724400 204 browser details YourSeq 71 1879 1967 3000 94.0% chr14 - 75972531 75972619 89 browser details YourSeq 62 2464 2527 3000 98.5% chr8 - 21456088 21456151 64 browser details YourSeq 62 2464 2527 3000 98.5% chr8 - 21594036 21594099 64 browser details YourSeq 62 2464 2527 3000 98.5% chr8 - 21703173 21703236 64 browser details YourSeq 58 2464 2527 3000 95.4% chr8 - 21095622 21095685 64 browser details YourSeq 58 2464 2527 3000 95.4% chr8 - 21326546 21326609 64 browser details YourSeq 23 1312 1334 3000 100.0% chr17 + 4660066 4660088 23 browser details YourSeq 21 735 755 3000 100.0% chr10 + 127819016 127819036 21

Note: The 3000 bp section upstream of Exon 7 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 - 6613366 6616365 3000 browser details YourSeq 462 1743 2987 3000 92.7% chr17 - 87403059 88054012 650954 browser details YourSeq 290 2621 3000 3000 92.9% chr7 - 46622951 46857421 234471 browser details YourSeq 288 2605 2996 3000 91.7% chr19 + 34925996 34926396 401 browser details YourSeq 254 2636 3000 3000 89.5% chr9 + 120683567 120683961 395 browser details YourSeq 248 2603 3000 3000 89.8% chr10 + 127486671 127487027 357 browser details YourSeq 242 2660 2996 3000 92.6% chr5 - 136558025 136558726 702 browser details YourSeq 238 2632 2996 3000 90.3% chr18 - 24517354 24517841 488 browser details YourSeq 235 2637 2999 3000 86.8% chr17 + 29827080 29827434 355 browser details YourSeq 221 2614 2996 3000 90.1% chr11 + 62588357 62588761 405 browser details YourSeq 218 2626 3000 3000 90.7% chr5 + 25584658 25585196 539 browser details YourSeq 215 2652 3000 3000 94.7% chr11 + 84216448 84217157 710 browser details YourSeq 208 2628 3000 3000 91.8% chr1 - 165239777 165240301 525 browser details YourSeq 201 2691 2996 3000 89.5% chr4 - 136308085 136308440 356 browser details YourSeq 201 2662 3000 3000 88.1% chr11 + 100449956 100450651 696 browser details YourSeq 192 2631 2996 3000 93.7% chr1 + 128246136 128246712 577 browser details YourSeq 182 2645 3000 3000 89.9% chr2 - 25187603 25188271 669 browser details YourSeq 182 2656 3000 3000 91.0% chr17 - 36993403 36993762 360 browser details YourSeq 180 2412 3000 3000 91.7% chr1 + 135362394 135363071 678 browser details YourSeq 179 2619 2996 3000 83.5% chr7 + 111022969 111023236 268

Note: The 3000 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tbrg4 transforming growth factor beta regulated gene 4 [ Mus musculus (house mouse) ] Gene ID: 21379, updated on 24-Oct-2019

Gene summary

Official Symbol Tbrg4 provided by MGI Official Full Name transforming growth factor beta regulated gene 4 provided by MGI Primary source MGI:MGI:1100868 See related Ensembl:ENSMUSG00000000384 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 Cpr2; Tb-12; R74877; AA120735; AA408001; AI527316; 2310042P22Rik Expression Ubiquitous expression in colon adult (RPKM 29.8), large intestine adult (RPKM 26.5) and 28 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 A1 See Tbrg4 in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (6615598..6626084, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (6515601..6526070, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 12 transcripts

Gene: Tbrg4 ENSMUSG00000000384

Description transforming growth factor beta regulated gene 4 [Source:MGI Symbol;Acc:MGI:1100868] Gene Synonyms 2310042P22Rik, Cpr2, TB-12 Location Chromosome 11: 6,615,598-6,626,067 reverse strand. GRCm38:CM001004.2 About this gene This gene has 12 transcripts (splice variants), 183 orthologues, 5 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tbrg4- ENSMUST00000189268.6 2347 630aa ENSMUSP00000140835.1 Protein coding CCDS24423 Q91YM4 TSL:1 212 GENCODE basic APPRIS P1

Tbrg4- ENSMUST00000000394.13 2290 630aa ENSMUSP00000000394.7 Protein coding CCDS24423 Q91YM4 TSL:1 201 GENCODE basic APPRIS P1

Tbrg4- ENSMUST00000136682.7 647 158aa ENSMUSP00000114174.1 Protein coding - Q5SWP1 CDS 3' 207 incomplete TSL:3

Tbrg4- ENSMUST00000144463.1 540 112aa ENSMUSP00000120103.1 Protein coding - Q5SWP0 CDS 3' 208 incomplete TSL:3

Tbrg4- ENSMUST00000156969.7 2324 630aa ENSMUSP00000114256.1 Nonsense mediated - Q91YM4 TSL:1 211 decay

Tbrg4- ENSMUST00000150697.7 2241 365aa ENSMUSP00000123131.1 Nonsense mediated - E9PUT1 TSL:1 209 decay

Tbrg4- ENSMUST00000134016.1 682 No - Retained intron - - TSL:3 206 protein

Tbrg4- ENSMUST00000151008.1 652 No - Retained intron - - TSL:1 210 protein

Tbrg4- ENSMUST00000132446.1 602 No - Retained intron - - TSL:2 205 protein

Tbrg4- ENSMUST00000131815.7 853 No - lncRNA - - TSL:3 204 protein

Tbrg4- ENSMUST00000131477.1 582 No - lncRNA - - TSL:5 203 protein

Tbrg4- ENSMUST00000131313.1 380 No - lncRNA - - TSL:3 202 protein

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30.47 kb Forward strand 6.61Mb 6.62Mb 6.63Mb Contigs AL603787.8 > Genes (Comprehensive set... < Nacad-201protein coding < Tbrg4-211nonsense mediated decay < Wap-201protein coding

< Tbrg4-212protein coding < Wap-203lncRNA

< Tbrg4-201protein coding < Wap-202lncRNA

< Tbrg4-209nonsense mediated decay

< Tbrg4-206retained intron < Tbrg4-204lncRNA

< Tbrg4-203lncRNA < Tbrg4-208protein coding

< Tbrg4-205retained intron

< Gm24313-201snoRNA

< Tbrg4-210retained intron

< Tbrg4-202lncRNA

< Snora5c-201snoRNA

< Tbrg4-207protein coding

Regulatory Build

6.61Mb 6.62Mb 6.63Mb Reverse strand 30.47 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

RNA gene processed transcript

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Transcript: ENSMUST00000189268

< Tbrg4-212protein coding

Reverse strand 10.47 kb

ENSMUSP00000140... Low complexity (Seg) Superfamily Armadillo-type fold SMART RAP domain Pfam FAST kinase-like protein, subdomain 2

FAST kinase leucine-rich RAP domain PROSITE profiles RAP domain PANTHER PTHR21228

PTHR21228:SF59

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 630

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

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