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Mouse Cnot10 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cnot10 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cnot10 gene (NCBI Reference Sequence: NM_153585 ; Ensembl: ENSMUSG00000056167 ) is located on Mouse chromosome 9. 20 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 19 (Transcript: ENSMUST00000070117). 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 Cnot10 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-307K14 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 12.54% 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: 2664 bp, and the size of intron 4 for 3'-loxP site insertion: 1471 bp. The size of effective cKO region: ~651 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 5 20 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cnot10 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(7151bp) | A(26.47% 1893) | C(20.19% 1444) | T(29.81% 2132) | G(23.52% 1682)

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% chr9 - 114629408 114632407 3000 browser details YourSeq 271 1367 2518 3000 88.4% chr6 + 119960359 120081301 120943 browser details YourSeq 229 1421 1953 3000 93.6% chr2 - 126715369 127072677 357309 browser details YourSeq 177 1404 1953 3000 82.7% chr7 + 99547367 99547607 241 browser details YourSeq 171 1293 1917 3000 82.3% chr1 + 176306851 176307123 273 browser details YourSeq 167 1335 1937 3000 85.0% chr16 - 32687659 32687927 269 browser details YourSeq 159 1773 1966 3000 93.5% chr11 + 70870756 70870956 201 browser details YourSeq 156 1773 1953 3000 92.0% chr14 + 45410936 45411112 177 browser details YourSeq 155 1677 1949 3000 92.4% chr9 - 114996476 114996875 400 browser details YourSeq 154 1778 1948 3000 94.7% chr3 - 74204231 74204400 170 browser details YourSeq 154 1578 1953 3000 84.0% chr11 - 72212729 72212953 225 browser details YourSeq 153 1771 1948 3000 95.9% chr2 - 29102396 29102575 180 browser details YourSeq 150 1775 1947 3000 96.4% chr11 - 109257322 109257532 211 browser details YourSeq 149 1366 1933 3000 77.8% chrX - 163621415 163621678 264 browser details YourSeq 148 1778 1953 3000 90.4% chr15 - 36529092 36529256 165 browser details YourSeq 146 1789 1949 3000 95.6% chr17 + 30561216 30561376 161 browser details YourSeq 145 1786 1950 3000 92.3% chr11 + 43629070 43629223 154 browser details YourSeq 144 1421 1946 3000 84.1% chr8 - 65214629 65214817 189 browser details YourSeq 144 1781 1953 3000 89.2% chr8 - 12973356 12973521 166 browser details YourSeq 143 1423 1945 3000 83.7% chr11 + 68878485 68878698 214

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% chr9 - 114625757 114628756 3000 browser details YourSeq 115 728 1051 3000 91.5% chr10 - 59582543 59925173 342631 browser details YourSeq 110 254 938 3000 87.1% chr9 + 114627819 114628503 685 browser details YourSeq 90 156 821 3000 93.3% chr7 - 92781557 93202191 420635 browser details YourSeq 80 981 1376 3000 76.5% chr3 - 143701235 143701319 85 browser details YourSeq 80 982 1065 3000 97.7% chr2 - 44562760 44562843 84 browser details YourSeq 80 984 1065 3000 98.8% chr5 + 110528275 110528356 82 browser details YourSeq 79 728 1065 3000 82.8% chr1 - 132174765 132175057 293 browser details YourSeq 79 982 1376 3000 76.2% chr17 + 42658913 42658996 84 browser details YourSeq 79 982 1065 3000 97.6% chr11 + 29113402 29113493 92 browser details YourSeq 79 984 1065 3000 98.8% chr1 + 45751760 45751842 83 browser details YourSeq 78 982 1065 3000 96.5% chr5 - 114237994 114238077 84 browser details YourSeq 78 982 1065 3000 96.5% chr11 + 46482391 46482474 84 browser details YourSeq 78 984 1065 3000 97.6% chr10 + 99616682 99616763 82 browser details YourSeq 78 983 1065 3000 97.6% chr1 + 171464906 171464990 85 browser details YourSeq 77 983 1065 3000 96.4% chrX - 74373902 74373984 83 browser details YourSeq 77 983 1065 3000 96.4% chr9 - 107011438 107011520 83 browser details YourSeq 77 983 1063 3000 97.6% chr8 - 68534431 68534511 81 browser details YourSeq 77 982 1066 3000 95.3% chr5 - 25505351 25505435 85 browser details YourSeq 77 983 1065 3000 96.4% chr6 + 77665625 77665707 83

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: Cnot10 CCR4-NOT transcription complex, subunit 10 [ Mus musculus (house mouse) ] Gene ID: 78893, updated on 12-Aug-2019

Gene summary

Official Symbol Cnot10 provided by MGI Official Full Name CCR4-NOT transcription complex, subunit 10 provided by MGI Primary source MGI:MGI:1926143 See related Ensembl:ENSMUSG00000056167 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 2600001P13Rik Expression Ubiquitous expression in limb E14.5 (RPKM 25.5), CNS E11.5 (RPKM 22.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 F3 See Cnot10 in Genome Data Viewer

Exon count: 19

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (114585874..114640220, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (114494996..114549192, complement)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 9 transcripts

Gene: Cnot10 ENSMUSG00000056167

Description CCR4-NOT transcription complex, subunit 10 [Source:MGI Symbol;Acc:MGI:1926143] Gene Synonyms 2600001P13Rik Location Chromosome 9: 114,585,878-114,640,184 reverse strand. GRCm38:CM001002.2 About this gene This gene has 9 transcripts (splice variants), 203 orthologues, 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

Cnot10- ENSMUST00000070117.7 2853 744aa ENSMUSP00000064840.6 Protein coding CCDS40794 Q8BH15 TSL:1 201 GENCODE basic APPRIS P1

Cnot10- ENSMUST00000216785.1 1723 409aa ENSMUSP00000149024.1 Protein coding - A0A1L1SQF2 TSL:5 206 GENCODE basic

Cnot10- ENSMUST00000238809.1 619 126aa ENSMUSP00000158898.1 Protein coding - - CDS 3' 209 incomplete

Cnot10- ENSMUST00000217148.1 3466 289aa ENSMUSP00000148963.1 Nonsense mediated - A0A1L1SQA7 TSL:1 207 decay

Cnot10- ENSMUST00000215155.1 3437 405aa ENSMUSP00000149745.1 Nonsense mediated - A0A1L1SS47 TSL:1 204 decay

Cnot10- ENSMUST00000213955.1 753 105aa ENSMUSP00000149867.1 Nonsense mediated - A0A1L1SSE2 TSL:1 203 decay

Cnot10- ENSMUST00000217296.1 4866 No - Retained intron - - TSL:1 208 protein

Cnot10- ENSMUST00000213539.1 1441 No - Retained intron - - TSL:1 202 protein

Cnot10- ENSMUST00000215701.1 487 No - lncRNA - - TSL:3 205 protein

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74.31 kb Forward strand

114.58Mb 114.60Mb 114.62Mb 114.64Mb Contigs < AC164109.2 AC161419.2 >

Genes < Cnot10-201protein coding (Comprehensive set...

< Cnot10-207nonsense mediated decay

< Cnot10-204nonsense mediated decay

< Cnot10-208retained intron

< Gm23232-201snRNA < Cnot10-206protein coding

< Cnot10-202retained intron

< Cnot10-203nonsense mediated decay

< Cnot10-205lncRNA

< Cnot10-209protein coding

< Gm7780-201processed pseudogene

Regulatory Build

114.58Mb 114.60Mb 114.62Mb 114.64Mb Reverse strand 74.31 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene pseudogene processed transcript

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Transcript: ENSMUST00000070117

< Cnot10-201protein coding

Reverse strand 54.20 kb

ENSMUSP00000064... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat PANTHER CCR4-NOT transcription complex subunit 10 Gene3D Tetratricopeptide-like helical domain superfamily

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 744

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

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