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Mouse Rb1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rb1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rb1 gene (NCBI Reference Sequence: NM_009029 ; Ensembl: ENSMUSG00000022105 ) is located on Mouse chromosome 14. 27 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 27 (Transcript: ENSMUST00000022701). Exon 8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rb1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-217E12 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: Homozygotes for targeted mutations exhibit abnormalities of the neuronal and hematopoietic systems and die in utero. Heterozygotes may develop pituitary tumors associated with loss of the normal allele.

Exon 8 starts from about 25.37% of the coding region. The knockout of Exon 8 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 2603 bp, and the size of intron 8 for 3'-loxP site insertion: 1630 bp. The size of effective cKO region: ~643 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 8 9 27 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rb1 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(7143bp) | A(31.39% 2242) | C(16.9% 1207) | T(31.43% 2245) | G(20.29% 1449)

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 - 73280515 73283514 3000 browser details YourSeq 144 859 1054 3000 87.2% chr17 + 10912133 10912334 202 browser details YourSeq 143 859 1054 3000 86.4% chr14 + 111965693 111965862 170 browser details YourSeq 137 880 1052 3000 88.2% chr16 - 27474605 27474774 170 browser details YourSeq 137 880 1053 3000 89.4% chr13 - 102679735 102679906 172 browser details YourSeq 136 1495 1783 3000 86.1% chr5 + 131407271 131407575 305 browser details YourSeq 130 880 1056 3000 87.9% chr10 + 7079969 7080143 175 browser details YourSeq 129 880 1053 3000 87.1% chr6 - 40621894 40622064 171 browser details YourSeq 128 880 1047 3000 87.4% chr11 - 12674398 12674561 164 browser details YourSeq 127 870 1056 3000 84.5% chr1 - 177102344 177102532 189 browser details YourSeq 127 881 1047 3000 86.6% chrX + 151197655 151197818 164 browser details YourSeq 125 897 1054 3000 94.4% chr2 - 136015165 136015340 176 browser details YourSeq 125 884 1057 3000 90.8% chr5 + 91845303 91845476 174 browser details YourSeq 125 859 1052 3000 89.9% chr15 + 43054302 43054497 196 browser details YourSeq 124 907 1055 3000 93.2% chr2 - 129371749 129761496 389748 browser details YourSeq 123 870 1054 3000 85.8% chr4 - 16155349 16155525 177 browser details YourSeq 123 881 1046 3000 85.8% chr12 + 109310140 109310302 163 browser details YourSeq 122 857 1056 3000 92.3% chr11 + 63822140 63822340 201 browser details YourSeq 122 880 1115 3000 84.1% chr10 + 12195268 12195467 200 browser details YourSeq 121 870 1052 3000 93.1% chr14 - 119316544 119316733 190

Note: The 3000 bp section upstream of Exon 8 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 - 73276872 73279871 3000 browser details YourSeq 183 1853 2378 3000 85.9% chr16 - 90579169 90579459 291 browser details YourSeq 182 1860 2366 3000 88.2% chr16 - 16308959 16610346 301388 browser details YourSeq 156 2194 2378 3000 93.6% chr13 + 86397817 86398006 190 browser details YourSeq 154 2202 2387 3000 91.8% chr6 + 79985886 79986070 185 browser details YourSeq 150 2213 2376 3000 96.4% chr17 - 54722888 54723331 444 browser details YourSeq 146 2201 2377 3000 93.8% chr12 - 3047591 3047773 183 browser details YourSeq 139 2201 2787 3000 82.4% chr1 - 15381573 15381793 221 browser details YourSeq 138 2209 2368 3000 92.5% chr4 + 71751924 71752082 159 browser details YourSeq 137 2202 2368 3000 95.5% chr3 - 133576030 133576205 176 browser details YourSeq 135 2206 2360 3000 94.8% chr17 - 28520356 28520510 155 browser details YourSeq 134 1841 2309 3000 87.5% chr2 - 139513671 139514244 574 browser details YourSeq 134 2200 2360 3000 94.2% chr4 + 73243123 73243290 168 browser details YourSeq 134 2202 2356 3000 91.4% chr2 + 87369006 87369157 152 browser details YourSeq 133 2202 2348 3000 93.9% chr4 - 139325457 139325602 146 browser details YourSeq 132 2199 2348 3000 95.3% chr18 + 60829255 60829784 530 browser details YourSeq 132 2202 2368 3000 94.3% chr12 + 18382445 18382617 173 browser details YourSeq 132 2210 2372 3000 91.3% chr10 + 56551387 56551564 178 browser details YourSeq 131 2206 2356 3000 94.0% chr11 - 80087999 80088163 165 browser details YourSeq 131 2201 2352 3000 91.4% chr10 - 49755646 49755795 150

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rb1 RB transcriptional corepressor 1 [ Mus musculus (house mouse) ] Gene ID: 19645, updated on 22-Oct-2019

Gene summary

Official Symbol Rb1 provided by MGI Official Full Name RB transcriptional corepressor 1 provided by MGI Primary source MGI:MGI:97874 See related Ensembl:ENSMUSG00000022105 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 Rb; pRb; Rb-1; pp105 Expression Ubiquitous expression in liver E14 (RPKM 8.6), whole brain E14.5 (RPKM 8.3) and 24 other tissues See more Orthologs human all

Genomic context

Location: 14 38.73 cM; 14 D3 See Rb1 in Genome Data Viewer

Exon count: 28

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (73192858..73325951, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (73595309..73725598, complement)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 6 transcripts

Gene: Rb1 ENSMUSG00000022105

Description RB transcriptional corepressor 1 [Source:MGI Symbol;Acc:MGI:97874] Gene Synonyms Rb, Rb-1, pRb, retinoblastoma 1 Location Chromosome 14: 73,183,673-73,325,822 reverse strand. GRCm38:CM001007.2 About this gene This gene has 6 transcripts (splice variants), 201 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 81 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rb1-201 ENSMUST00000022701.6 4656 921aa ENSMUSP00000022701.6 Protein coding CCDS27267 P13405 TSL:1 GENCODE basic APPRIS P1

Rb1-203 ENSMUST00000164624.1 324 47aa ENSMUSP00000125967.1 Nonsense mediated decay - E9Q5C5 TSL:5

Rb1-204 ENSMUST00000168495.1 946 No protein - Retained intron - - TSL:2

Rb1-205 ENSMUST00000169002.1 355 No protein - Retained intron - - TSL:3

Rb1-202 ENSMUST00000163932.1 411 No protein - lncRNA - - TSL:5

Rb1-206 ENSMUST00000170967.1 408 No protein - lncRNA - - TSL:5

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162.15 kb Forward strand 73.20Mb 73.25Mb 73.30Mb Genes Rcbtb2-209 >protein coding Lpar6-201 >protein coding Gm49131-201 >processed pseudogene (Comprehensive set...

Rcbtb2-226 >protein coding

Rcbtb2-223 >protein coding

Rcbtb2-217 >protein coding

Rcbtb2-219 >protein coding

Rcbtb2-201 >protein coding

Rcbtb2-202 >protein coding

Rcbtb2-218 >protein coding

Rcbtb2-207 >nonsense mediated decay

Rcbtb2-208 >nonsense mediated decay

Rcbtb2-215 >protein coding

Rcbtb2-220 >retained intron

Contigs CT571265.12 > AC154718.2 > Genes (Comprehensive set... < Gm17233-201lncRNA < Rb1-201protein coding

< Rb1-202lncRNA

< Rb1-204retained intron < Rb1-206lncRNA < Rb1-205retained intron

< Mir687-201miRNA < Rb1-203nonsense mediated decay

Regulatory Build

73.20Mb 73.25Mb 73.30Mb Reverse strand 162.15 kb

Regulation Legend CTCF Enhancer 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 pseudogene

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Transcript: ENSMUST00000022701

< Rb1-201protein coding

Reverse strand 130.32 kb

ENSMUSP00000022... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Cyclin-like superfamily SMART Retinoblastoma-associated protein, A-box Retinoblastoma-associated protein, C-terminal

Retinoblastoma-associated protein, N-terminal Cyclin-like Pfam Retinoblastoma-associated protein, A-box Retinoblastoma-associated protein, C-terminal

Retinoblastoma-associated protein, N-terminal Retinoblastoma-associated protein, B-box PANTHER Retinoblastoma protein family

Retinoblastoma-associated protein Gene3D 1.10.472.140 1.10.472.10 CDD Cyclin-like

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 921

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

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