https://www.alphaknockout.com

Mouse Rbbp8 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rbbp8 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rbbp8 gene (NCBI Reference Sequence: NM_001252495 ; Ensembl: ENSMUSG00000041238 ) is located on Mouse chromosome 18. 19 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 19 (Transcript: ENSMUST00000115861). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rbbp8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-122B4 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: Embryos homozygous for a knock-out allele die at E4.0 as blastocysts fail to enter S phase and arrest at G1,leading to elevated cell death. Heterozygous mutant mice display a shortened lifespan due to formation of multiple tumors, mostly large lymphomasof both B and T cells.

Exon 6 starts from about 13.55% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 3618 bp, and the size of intron 6 for 3'-loxP site insertion: 8882 bp. The size of effective cKO region: ~567 bp. The cKO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 6 19 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rbbp8 Homology arm cKO region loxP site

Page 2 of 8 https://www.alphaknockout.com

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(7067bp) | A(31.53% 2228) | C(18.3% 1293) | T(31.58% 2232) | G(18.59% 1314)

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.

Page 3 of 8 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr18 + 11693494 11696493 3000 browser details YourSeq 285 889 1581 3000 86.3% chr16 + 55766216 55767000 785 browser details YourSeq 281 814 1593 3000 81.3% chr5 - 64670767 64671637 871 browser details YourSeq 279 740 1205 3000 86.5% chr7 - 102374013 102374567 555 browser details YourSeq 268 1894 2624 3000 83.7% chr9 + 15693026 15693871 846 browser details YourSeq 261 1042 2347 3000 89.1% chr10 - 8046974 8048309 1336 browser details YourSeq 249 1008 1862 3000 87.2% chr16 - 13485415 13486295 881 browser details YourSeq 248 1002 1735 3000 85.8% chr1 - 62890466 62891217 752 browser details YourSeq 247 837 1294 3000 86.4% chr3 - 82889634 82890143 510 browser details YourSeq 246 1688 2375 3000 87.8% chr4 + 122917754 122918651 898 browser details YourSeq 227 1367 1962 3000 78.9% chr5 - 66735875 66736444 570 browser details YourSeq 220 999 2543 3000 81.2% chr16 - 12190267 12191514 1248 browser details YourSeq 220 863 1472 3000 86.3% chrX + 48617133 48618063 931 browser details YourSeq 217 1053 1772 3000 86.6% chr1 - 150559478 150560209 732 browser details YourSeq 217 1042 1593 3000 89.4% chr16 + 20795492 20796069 578 browser details YourSeq 214 955 1777 3000 83.3% chrX - 75072285 75073043 759 browser details YourSeq 206 824 1610 3000 85.4% chr7 - 68655454 68656277 824 browser details YourSeq 202 824 1709 3000 88.9% chr19 + 20892372 20893306 935 browser details YourSeq 201 740 1487 3000 85.3% chr15 + 71344606 71345390 785 browser details YourSeq 200 1850 2598 3000 80.3% chr2 + 179998686 179999185 500

Note: The 3000 bp section upstream of Exon 6 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% chr18 + 11697061 11700060 3000 browser details YourSeq 199 2114 2443 3000 95.4% chrX + 48472129 48472646 518 browser details YourSeq 194 2103 2566 3000 91.9% chr2 + 34789361 34789806 446 browser details YourSeq 193 2114 2312 3000 98.5% chr13 + 9353389 9353587 199 browser details YourSeq 190 2115 2312 3000 98.0% chr5 + 144994614 144994811 198 browser details YourSeq 189 2114 2315 3000 95.5% chr5 - 144745959 144746158 200 browser details YourSeq 188 2114 2446 3000 93.5% chr4 - 152167413 152167908 496 browser details YourSeq 187 2114 2313 3000 95.5% chr15 - 8814003 8814200 198 browser details YourSeq 187 2113 2312 3000 95.5% chr14 + 61247694 61247891 198 browser details YourSeq 186 2114 2312 3000 95.5% chr5 - 139950801 139950997 197 browser details YourSeq 186 2114 2312 3000 95.5% chr16 - 29901462 29901658 197 browser details YourSeq 184 2114 2444 3000 87.8% chr9 - 111876051 111876282 232 browser details YourSeq 184 2118 2358 3000 93.8% chr6 - 29840702 29841085 384 browser details YourSeq 184 2114 2312 3000 95.0% chr11 - 80254021 80254217 197 browser details YourSeq 184 2114 2370 3000 93.0% chr14 + 48325190 48719077 393888 browser details YourSeq 183 2114 2312 3000 96.5% chr17 - 6384634 6705830 321197 browser details YourSeq 182 2114 2312 3000 94.5% chr9 - 57648975 57649171 197 browser details YourSeq 182 2114 2312 3000 94.5% chr5 + 150714880 150715076 197 browser details YourSeq 182 2114 2312 3000 94.5% chr3 + 70928629 70928825 197 browser details YourSeq 179 2056 2312 3000 92.8% chrX - 36822935 36823228 294

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

Page 4 of 8 https://www.alphaknockout.com

Gene and protein information: Rbbp8 retinoblastoma binding protein 8, endonuclease [ Mus musculus (house mouse) ] Gene ID: 225182, updated on 12-Aug-2019

Gene summary

Official Symbol Rbbp8 provided by MGI Official Full Name retinoblastoma binding protein 8, endonuclease provided by MGI Primary source MGI:MGI:2442995 See related Ensembl:ENSMUSG00000041238 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 RIM; CtIP; SAE2; RBBP-8; 9930104E21Rik Expression Broad expression in CNS E11.5 (RPKM 6.2), placenta adult (RPKM 5.1) and 23 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 A1 See Rbbp8 in Genome Data Viewer

Exon count: 21

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (11633276..11743207)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (11816351..11901716)

Chromosome 18 - NC_000084.6

Page 5 of 8 https://www.alphaknockout.com

Transcript information: This gene has 11 transcripts

Gene: Rbbp8 ENSMUSG00000041238

Description retinoblastoma binding protein 8, endonuclease [Source:MGI Symbol;Acc:MGI:2442995] Gene Synonyms 9930104E21Rik, CtIP Location Chromosome 18: 11,633,276-11,745,221 forward strand. GRCm38:CM001011.2 About this gene This gene has 11 transcripts (splice variants), 157 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rbbp8- ENSMUST00000115861.8 3523 893aa ENSMUSP00000111527.2 Protein coding CCDS37738 Q80YR6 TSL:1 202 GENCODE basic APPRIS P1

Rbbp8- ENSMUST00000047322.7 3209 893aa ENSMUSP00000046255.6 Protein coding CCDS37738 Q80YR6 TSL:1 201 GENCODE basic APPRIS P1

Rbbp8- ENSMUST00000234499.1 586 118aa ENSMUSP00000157079.1 Protein coding - A0A3Q4L2U0 CDS 3' 206 incomplete

Rbbp8- ENSMUST00000234984.1 5471 612aa ENSMUSP00000157008.1 Nonsense mediated - A0A3Q4EGK0 - 210 decay

Rbbp8- ENSMUST00000235039.1 3360 247aa ENSMUSP00000157244.1 Nonsense mediated - A0A3Q4EGL1 - 211 decay

Rbbp8- ENSMUST00000234616.1 781 26aa ENSMUSP00000157098.1 Nonsense mediated - A0A3Q4EBX2 CDS 5' 207 decay incomplete

Rbbp8- ENSMUST00000234184.1 3714 No - Retained intron - - - 205 protein

Rbbp8- ENSMUST00000234074.1 1904 No - Retained intron - - - 203 protein

Rbbp8- ENSMUST00000234766.1 1736 No - Retained intron - - - 209 protein

Rbbp8- ENSMUST00000234744.1 857 No - Retained intron - - - 208 protein

Rbbp8- ENSMUST00000234161.1 679 No - Retained intron - - - 204 protein

Page 6 of 8 https://www.alphaknockout.com

131.95 kb Forward strand 11.64Mb 11.66Mb 11.68Mb 11.70Mb 11.72Mb 11.74Mb Genes (Comprehensive set... Gm50067-201 >processed pseudogene Rbbp8-210 >nonsense mediated decay

Rbbp8-202 >protein coding

Rbbp8-211 >nonsense mediated decay

Rbbp8-205 >retained intron Rbbp8-204 >retained intron

Rbbp8-203 >retained intron Rbbp8-209 >retained intron

Rbbp8-201 >protein coding

Rbbp8-208 >retained intron

Rbbp8-206 >protein coding

Rbbp8-207 >nonsense mediated decay

Contigs AC090479.6 > < AC115894.6 Genes < Gm50069-201lncRNA < Gm18713-201processed pseudogene (Comprehensive set...

Regulatory Build

11.64Mb 11.66Mb 11.68Mb 11.70Mb 11.72Mb 11.74Mb Reverse strand 131.95 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

pseudogene RNA gene processed transcript

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000115861

109.93 kb Forward strand

Rbbp8-202 >protein coding

ENSMUSP00000111... MobiDB lite Coiled-coils (Ncoils) Pfam DNA endonuclease Ctp1, N-terminal DNA endonuclease Ctp1, C-terminal

PANTHER DNA endonuclease RBBP8

DNA endonuclease RBBP8-like

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 893

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

Page 8 of 8