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Mouse Rcbtb1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rcbtb1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rcbtb1 gene (NCBI Reference Sequence: NM_027764 ; Ensembl: ENSMUSG00000035469 ) is located on Mouse chromosome 14. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000043227). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rcbtb1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-193F7 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 4 starts from about 17.45% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 7010 bp, and the size of intron 4 for 3'-loxP site insertion: 3654 bp. The size of effective cKO region: ~667 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 4 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rcbtb1 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(7167bp) | A(25.0% 1792) | C(20.73% 1486) | T(32.73% 2346) | G(21.53% 1543)

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% chr14 + 59214347 59217346 3000 browser details YourSeq 160 971 2095 3000 94.0% chr14 - 59215317 59216441 1125 browser details YourSeq 127 2091 2258 3000 92.7% chr4 - 94611641 94611817 177 browser details YourSeq 127 2109 2258 3000 94.5% chr10 - 119181798 119181967 170 browser details YourSeq 126 2109 2270 3000 91.0% chr1 - 193392810 193392995 186 browser details YourSeq 121 2109 2258 3000 92.5% chr19 + 23308754 23308926 173 browser details YourSeq 119 2088 2219 3000 96.3% chr15 - 79470111 79470252 142 browser details YourSeq 118 2110 2261 3000 90.5% chr12 - 101943314 101943479 166 browser details YourSeq 118 2093 2240 3000 90.8% chr13 + 3609107 3609253 147 browser details YourSeq 118 2109 2466 3000 92.2% chr10 + 125797918 125798504 587 browser details YourSeq 118 2109 2248 3000 92.9% chr1 + 192199977 192200126 150 browser details YourSeq 115 2109 2244 3000 94.7% chr1 - 185399590 185399737 148 browser details YourSeq 115 2109 2238 3000 94.7% chr7 + 141255163 141255293 131 browser details YourSeq 115 2109 2238 3000 94.7% chr3 + 116697200 116697330 131 browser details YourSeq 115 2109 2238 3000 94.7% chr3 + 84330677 84330807 131 browser details YourSeq 115 2109 2238 3000 94.7% chr19 + 29746629 29746759 131 browser details YourSeq 114 2109 2240 3000 94.0% chr12 + 57562824 57562965 142 browser details YourSeq 113 2109 2238 3000 93.9% chr18 - 35760096 35760226 131 browser details YourSeq 112 1645 2219 3000 81.6% chr11 - 100418788 100419299 512 browser details YourSeq 111 2109 2238 3000 93.1% chr2 + 30204475 30204605 131

Note: The 3000 bp section upstream of Exon 4 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% chr14 + 59218014 59221013 3000 browser details YourSeq 126 1927 2989 3000 95.0% chr14 - 59219940 59221002 1063 browser details YourSeq 65 2395 2573 3000 97.2% chr7 + 4508126 4508610 485 browser details YourSeq 62 2237 2445 3000 95.6% chr10 + 114832745 114832957 213 browser details YourSeq 61 2374 2445 3000 97.0% chr11 + 81903150 81903228 79 browser details YourSeq 61 2392 2485 3000 98.5% chr10 + 60380683 60380779 97 browser details YourSeq 58 2395 2454 3000 98.4% chr3 - 31766047 31766106 60 browser details YourSeq 56 2395 2453 3000 100.0% chr3 - 153068200 153068283 84 browser details YourSeq 56 2392 2447 3000 100.0% chr16 - 6123104 6123159 56 browser details YourSeq 54 2392 2445 3000 100.0% chr14 - 78285903 78285956 54 browser details YourSeq 54 2392 2445 3000 100.0% chr14 - 23919376 23919429 54 browser details YourSeq 54 2392 2445 3000 100.0% chr10 + 23466131 23466184 54 browser details YourSeq 54 2396 2452 3000 98.3% chr1 + 72522108 72522166 59 browser details YourSeq 53 2392 2445 3000 100.0% chr18 - 43316827 43316902 76 browser details YourSeq 52 2394 2445 3000 100.0% chr13 - 13575595 13575646 52 browser details YourSeq 52 2392 2445 3000 98.2% chr19 + 58196830 58196883 54 browser details YourSeq 52 2392 2445 3000 98.2% chr10 + 18414525 18414578 54 browser details YourSeq 51 2395 2445 3000 100.0% chr8 - 82683911 82683961 51 browser details YourSeq 51 2395 2445 3000 100.0% chr6 - 79539201 79539251 51 browser details YourSeq 51 2395 2445 3000 100.0% chr2 - 162485763 162485813 51

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: Rcbtb1 regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1 [ Mus musculus (house mouse) ] Gene ID: 71330, updated on 24-Oct-2019

Gene summary

Official Symbol Rcbtb1 provided by MGI Official Full Name regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1 provided by MGI Primary source MGI:MGI:1918580 See related Ensembl:ENSMUSG00000035469 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 CLLD7; CLLL7; AW111883; 5430409I18Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 17.7), CNS E14 (RPKM 14.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 C3 See Rcbtb1 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (59201018..59237267)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (59820065..59856102)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 11 transcripts

Gene: Rcbtb1 ENSMUSG00000035469

Description regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1 [Source:MGI Symbol;Acc:MGI:1918580] Gene Synonyms 5430409I18Rik Location Chromosome 14: 59,201,209-59,237,265 forward strand. GRCm38:CM001007.2 About this gene This gene has 11 transcripts (splice variants), 254 orthologues, 9 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rcbtb1- ENSMUST00000022551.13 3957 531aa ENSMUSP00000022551.7 Protein coding CCDS27168 A0A0R4J025 TSL:1 201 GENCODE basic APPRIS P1

Rcbtb1- ENSMUST00000043227.12 3903 531aa ENSMUSP00000037030.6 Protein coding CCDS27168 A0A0R4J025 TSL:1 202 GENCODE basic APPRIS P1

Rcbtb1- ENSMUST00000174830.1 852 94aa ENSMUSP00000133421.1 Protein coding - G3UWU1 CDS 5' 211 incomplete TSL:5

Rcbtb1- ENSMUST00000173547.7 765 208aa ENSMUSP00000134360.1 Protein coding - G3UZ62 CDS 3' 209 incomplete TSL:5

Rcbtb1- ENSMUST00000142326.1 447 33aa ENSMUSP00000134542.1 Protein coding - G3UZL2 CDS 3' 205 incomplete TSL:2

Rcbtb1- ENSMUST00000140136.8 421 1aa ENSMUSP00000134515.1 Protein coding - - CDS 3' 204 incomplete TSL:3

Rcbtb1- ENSMUST00000174009.7 2163 40aa ENSMUSP00000133369.1 Nonsense mediated - G3UYZ5 TSL:1 210 decay

Rcbtb1- ENSMUST00000172810.1 899 40aa ENSMUSP00000134284.1 Nonsense mediated - G3UYZ5 TSL:5 208 decay

Rcbtb1- ENSMUST00000147280.1 765 No - Retained intron - - TSL:3 206 protein

Rcbtb1- ENSMUST00000095778.7 1499 No - lncRNA - - TSL:1 203 protein

Rcbtb1- ENSMUST00000153225.1 754 No - lncRNA - - TSL:3 207 protein

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56.06 kb Forward strand

59.20Mb 59.21Mb 59.22Mb 59.23Mb 59.24Mb Genes (Comprehensive set... Rcbtb1-209 >protein coding

Rcbtb1-202 >protein coding

Rcbtb1-210 >nonsense mediated decay

Rcbtb1-201 >protein coding

Rcbtb1-204 >protein coding Rcbtb1-207 >lncRNA

Rcbtb1-203 >lncRNA Rcbtb1-206 >retained intron

Rcbtb1-205 >protein coding Rcbtb1-211 >protein coding

Rcbtb1-208 >nonsense mediated decay

Contigs AC166169.2 > Genes < Phf11-ps-201unprocessed pseudogene (Comprehensive set...

Regulatory Build

59.20Mb 59.21Mb 59.22Mb 59.23Mb 59.24Mb Reverse strand 56.06 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000043227

36.04 kb Forward strand

Rcbtb1-202 >protein coding

protein_pic

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

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