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Mouse Cnot9 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cnot9 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cnot9 gene (NCBI Reference Sequence: NM_021383.5 ; Ensembl: ENSMUSG00000026174 ) is located on Mouse chromosome 1. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000087215). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cnot9 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-70H11 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 an ENU-induced allele exhibit abnormal nervous system and brain morphology, cranioedema, and caudal body truncation. Mice carrying a targeted allele exhibit embryonic lethality.

Exon 2 starts from about 2.79% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 10608 bp, and the size of intron 3 for 3'-loxP site insertion: 3301 bp. The size of effective cKO region: ~2447 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 2 3 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cnot9 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(8947bp) | A(26.96% 2412) | C(20.79% 1860) | T(31.89% 2853) | G(20.36% 1822)

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% chr1 + 74513821 74516820 3000 browser details YourSeq 734 1 1120 3000 87.5% chr16 + 93633497 93634664 1168 browser details YourSeq 729 36 1120 3000 87.4% chr17 - 87028724 87029851 1128 browser details YourSeq 714 30 1126 3000 87.4% chr4 - 40282699 40283850 1152 browser details YourSeq 714 1 1120 3000 85.8% chrX + 77492127 77493301 1175 browser details YourSeq 712 35 1120 3000 87.3% chr6 - 82248138 82249275 1138 browser details YourSeq 711 1 1120 3000 87.1% chr6 + 50512797 50513966 1170 browser details YourSeq 710 74 1120 3000 86.8% chrX - 19676211 19677297 1087 browser details YourSeq 707 65 1120 3000 86.1% chr6 - 29097040 29098144 1105 browser details YourSeq 707 1 1120 3000 87.4% chr15 - 4964621 4965779 1159 browser details YourSeq 706 35 1116 3000 87.3% chr13 - 26620877 26622010 1134 browser details YourSeq 705 35 1120 3000 85.6% chr4 + 34591097 34592229 1133 browser details YourSeq 704 65 1120 3000 87.7% chr12 - 30660699 30661807 1109 browser details YourSeq 703 1 1120 3000 86.4% chr7 + 116772883 116774039 1157 browser details YourSeq 703 1 1120 3000 88.3% chr17 + 5380900 5382062 1163 browser details YourSeq 701 1 1120 3000 87.0% chr4 + 57367915 57369080 1166 browser details YourSeq 698 65 1120 3000 88.0% chr11 - 9380923 9382017 1095 browser details YourSeq 697 65 1120 3000 86.2% chr4 - 50365948 50367049 1102 browser details YourSeq 697 1 1120 3000 85.6% chr18 + 28914442 28915621 1180 browser details YourSeq 695 1 1120 3000 89.4% chr11 - 24582425 24583598 1174

Note: The 3000 bp section upstream of Exon 2 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% chr1 + 74519268 74522267 3000 browser details YourSeq 247 958 1279 3000 89.9% chr15 + 94014782 94240931 226150 browser details YourSeq 246 841 1284 3000 85.8% chr19 - 31645510 31645950 441 browser details YourSeq 242 940 1284 3000 87.0% chr18 - 88172793 88173137 345 browser details YourSeq 240 913 1286 3000 84.4% chr12 - 11630564 11630900 337 browser details YourSeq 239 953 1284 3000 89.5% chr18 - 48027975 48028314 340 browser details YourSeq 238 952 1284 3000 85.3% chr19 - 47915972 47916294 323 browser details YourSeq 237 942 1284 3000 85.3% chr13 - 64847147 64847470 324 browser details YourSeq 235 940 1275 3000 87.9% chr9 - 48009461 48009814 354 browser details YourSeq 235 952 1284 3000 86.8% chr19 - 53225021 53225347 327 browser details YourSeq 235 941 1284 3000 83.9% chr17 - 63216334 63216671 338 browser details YourSeq 235 945 1284 3000 90.4% chr17 + 75231119 75231506 388 browser details YourSeq 233 964 1274 3000 90.6% chr6 - 30702299 30702612 314 browser details YourSeq 232 961 1284 3000 88.1% chrX - 51298399 51298717 319 browser details YourSeq 231 944 1263 3000 89.1% chr3 - 133047548 133047863 316 browser details YourSeq 230 944 1284 3000 87.0% chr7 - 42524147 42524483 337 browser details YourSeq 230 960 1284 3000 89.1% chrX + 159478000 159478319 320 browser details YourSeq 229 961 1275 3000 87.4% chr16 - 87081357 87081666 310 browser details YourSeq 228 939 1284 3000 87.9% chr2 - 90055223 90055562 340 browser details YourSeq 228 939 1284 3000 89.1% chr14 - 38030346 38030695 350

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cnot9 CCR4-NOT transcription complex, subunit 9 [ Mus musculus (house mouse) ] Gene ID: 58184, updated on 26-Jun-2020

Gene summary

Official Symbol Cnot9 provided by MGI Official Full Name CCR4-NOT transcription complex, subunit 9 provided by MGI Primary source MGI:MGI:1928902 See related Ensembl:ENSMUSG00000026174 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 Fl10; Rqcd1; AI593551; 2610007F23Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 22.1), limb E14.5 (RPKM 19.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 C3 See Cnot9 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (74506036..74530842)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (74552634..74577416)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 3 transcripts

Gene: Cnot9 ENSMUSG00000026174

Description CCR4-NOT transcription complex, subunit 9 [Source:MGI Symbol;Acc:MGI:1928902] Gene Synonyms 2610007F23Rik, FL10, Rqcd1 Location Chromosome 1: 74,506,058-74,530,842 forward strand. GRCm38:CM000994.2 About this gene This gene has 3 transcripts (splice variants), 309 orthologues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cnot9-201 ENSMUST00000087215.6 3268 299aa ENSMUSP00000084466.5 Protein coding CCDS35615 Q9JKY0 TSL:1 GENCODE basic APPRIS P1

Cnot9-202 ENSMUST00000137100.1 831 No protein - Processed transcript - - TSL:3

Cnot9-203 ENSMUST00000147810.1 772 No protein - Retained intron - - TSL:3

44.78 kb Forward strand 74.50Mb 74.51Mb 74.52Mb 74.53Mb 74.54Mb Genes (Comprehensive set... Cnot9-201 >protein coding

Cnot9-202 >processed transcript

Cnot9-203 >retained intron

Contigs AC117610.7 > Genes < Usp37-201protein coding (Comprehensive set...

< Usp37-205protein coding

< Usp37-207retained intron

< Usp37-202protein coding

< Usp37-206retained intron

< Usp37-203processed transcript

< Gm37733-201TEC

Regulatory Build

74.50Mb 74.51Mb 74.52Mb 74.53Mb 74.54Mb Reverse strand 44.78 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

processed transcript

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Transcript: ENSMUST00000087215

24.79 kb Forward strand

Cnot9-201 >protein coding

ENSMUSP00000084... Low complexity (Seg) Superfamily Armadillo-type fold Pfam PF04078 PANTHER PTHR12262

CCR4-NOT transcription complex subunit 9 Gene3D Armadillo-like helical

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

Variant Legend

splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 299

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

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