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Mouse Cnot7 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cnot7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cnot7 gene (NCBI Reference Sequence: NM_011135 ; Ensembl: ENSMUSG00000031601 ) is located on Mouse chromosome 8. 7 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000034012). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cnot7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-380N5 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: Homozygous null mice display male sterility with oligo-teratozoospermia, impaired sperm motility, unsynchronized spermatid maturation, and Sertoli cell abnormalities.

Exon 3 starts from about 13.8% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 2366 bp, and the size of intron 3 for 3'-loxP site insertion: 6541 bp. The size of effective cKO region: ~694 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 3 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cnot7 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(7194bp) | A(27.47% 1976) | C(17.04% 1226) | T(34.31% 2468) | G(21.18% 1524)

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% chr8 - 40507899 40510898 3000 browser details YourSeq 183 972 1436 3000 96.0% chr11 - 94380363 94380841 479 browser details YourSeq 155 1079 1436 3000 92.8% chr7 + 111143400 111143810 411 browser details YourSeq 148 1277 1436 3000 96.9% chr11 + 120842265 121234600 392336 browser details YourSeq 146 940 1435 3000 91.6% chr10 + 4666507 4667074 568 browser details YourSeq 145 1264 1437 3000 89.3% chr14 - 27033701 27033868 168 browser details YourSeq 144 1278 1437 3000 96.8% chr1 - 53511308 53511483 176 browser details YourSeq 144 1276 1437 3000 92.5% chr10 + 80275840 80275998 159 browser details YourSeq 144 1279 1437 3000 93.7% chr1 + 82818505 82818661 157 browser details YourSeq 143 1278 1437 3000 93.1% chr2 + 153819362 153819519 158 browser details YourSeq 143 1282 1437 3000 94.2% chr11 + 6625478 6625631 154 browser details YourSeq 143 1277 1440 3000 94.4% chr1 + 58467144 58467306 163 browser details YourSeq 142 1259 1440 3000 92.3% chr16 - 29543499 29543681 183 browser details YourSeq 142 1286 1436 3000 95.4% chr15 - 51932842 51932990 149 browser details YourSeq 142 1275 1437 3000 92.0% chr17 + 26426510 26426670 161 browser details YourSeq 141 1282 1438 3000 94.2% chr11 - 5004677 5004832 156 browser details YourSeq 141 1279 1441 3000 94.5% chr2 + 149969586 149969761 176 browser details YourSeq 140 1260 1436 3000 92.3% chr6 - 26817977 26818164 188 browser details YourSeq 140 1102 1431 3000 93.3% chr4 - 148520127 148520619 493 browser details YourSeq 140 1269 1437 3000 89.9% chr14 - 45869259 45869425 167

Note: The 3000 bp section upstream of Exon 3 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% chr8 - 40504205 40507204 3000 browser details YourSeq 105 68 237 3000 78.7% chr17 + 74482654 74482807 154 browser details YourSeq 94 60 211 3000 86.9% chr1 + 59753869 59754468 600 browser details YourSeq 83 109 212 3000 86.2% chr19 - 10405969 10406069 101 browser details YourSeq 81 68 198 3000 87.1% chr5 - 77200677 77200809 133 browser details YourSeq 80 72 211 3000 81.5% chr1 + 31512060 31512195 136 browser details YourSeq 78 68 177 3000 83.9% chrX - 100471084 100471191 108 browser details YourSeq 78 68 196 3000 90.7% chr4 + 130645730 130646291 562 browser details YourSeq 75 115 212 3000 85.2% chr7 + 35337690 35337784 95 browser details YourSeq 74 102 212 3000 91.1% chr16 + 55817220 55817330 111 browser details YourSeq 72 68 196 3000 92.9% chr7 + 105644101 105644229 129 browser details YourSeq 71 102 214 3000 81.4% chr7 - 4747846 4747956 111 browser details YourSeq 71 68 188 3000 78.8% chr6 - 122511634 122511753 120 browser details YourSeq 71 77 194 3000 86.9% chr18 + 60488743 60488858 116 browser details YourSeq 71 80 201 3000 76.7% chr12 + 76267965 76268084 120 browser details YourSeq 71 71 188 3000 88.9% chr1 + 170930906 171004258 73353 browser details YourSeq 68 116 214 3000 85.3% chr11 - 103229100 103229199 100 browser details YourSeq 68 109 209 3000 83.4% chr12 + 85224059 85224158 100 browser details YourSeq 67 77 209 3000 85.9% chr1 - 127866760 127866892 133 browser details YourSeq 67 62 214 3000 81.2% chr10 + 58669867 58670011 145

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: Cnot7 CCR4-NOT transcription complex, subunit 7 [ Mus musculus (house mouse) ] Gene ID: 18983, updated on 24-Oct-2019

Gene summary

Official Symbol Cnot7 provided by MGI Official Full Name CCR4-NOT transcription complex, subunit 7 provided by MGI Primary source MGI:MGI:1298230 See related Ensembl:ENSMUSG00000031601 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 Caf1; Pop2; CAF-1; AU022737 Expression Broad expression in CNS E14 (RPKM 22.0), CNS E18 (RPKM 21.1) and 23 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 A4 See Cnot7 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (40492538..40515847, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (41578390..41596658, complement)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 11 transcripts

Gene: Cnot7 ENSMUSG00000031601

Description CCR4-NOT transcription complex, subunit 7 [Source:MGI Symbol;Acc:MGI:1298230] Gene Synonyms Caf1 Location Chromosome 8: 40,492,540-40,515,847 reverse strand. GRCm38:CM001001.2 About this gene This gene has 11 transcripts (splice variants), 235 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 21 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cnot7-211 ENSMUST00000149992.1 2982 285aa ENSMUSP00000117304.1 Protein coding CCDS22253 Q543X5 Q60809 TSL:1 GENCODE basic APPRIS P1

Cnot7-201 ENSMUST00000034012.9 2615 285aa ENSMUSP00000034012.3 Protein coding CCDS22253 Q543X5 Q60809 TSL:1 GENCODE basic APPRIS P1

Cnot7-203 ENSMUST00000132032.7 2542 285aa ENSMUSP00000122933.1 Protein coding CCDS22253 Q543X5 Q60809 TSL:1 GENCODE basic APPRIS P1

Cnot7-206 ENSMUST00000135269.7 2461 248aa ENSMUSP00000119319.1 Protein coding CCDS72114 Q3TLK9 TSL:1 GENCODE basic

Cnot7-209 ENSMUST00000144970.7 3888 No protein - Retained intron - - TSL:2

Cnot7-205 ENSMUST00000132740.1 840 No protein - Retained intron - - TSL:2

Cnot7-210 ENSMUST00000146280.1 726 No protein - lncRNA - - TSL:1

Cnot7-208 ENSMUST00000142455.7 679 No protein - lncRNA - - TSL:2

Cnot7-202 ENSMUST00000128237.1 484 No protein - lncRNA - - TSL:3

Cnot7-207 ENSMUST00000139558.1 406 No protein - lncRNA - - TSL:5

Cnot7-204 ENSMUST00000132200.1 312 No protein - lncRNA - - TSL:3

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43.31 kb Forward strand

40.49Mb 40.50Mb 40.51Mb 40.52Mb Genes Zdhhc2-201 >protein coding Zdhhc2-204 >lncRNA Vps37a-201 >protein coding (Comprehensive set...

Zdhhc2-202 >protein coding Vps37a-204 >retained intron

Zdhhc2-205 >protein coding Vps37a-202 >retained intron

Vps37a-206 >retained intron

Vps37a-203 >protein coding

Vps37a-205 >lncRNA

Contigs AC118012.9 > Genes (Comprehensive set... < Cnot7-201protein coding

< Cnot7-203protein coding

< Cnot7-209retained intron

< Cnot7-206protein coding

< Cnot7-211protein coding

< Cnot7-208lncRNA < Cnot7-210lncRNA

< Cnot7-205retained intron < Cnot7-202lncRNA

< Cnot7-204lncRNA < Cnot7-207lncRNA

Regulatory Build

40.49Mb 40.50Mb 40.51Mb 40.52Mb Reverse strand 43.31 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000034012

< Cnot7-201protein coding

Reverse strand 19.21 kb

ENSMUSP00000034... Low complexity (Seg) Superfamily Ribonuclease H-like superfamily

Pfam Ribonuclease CAF1 PANTHER CCR4-NOT transcription complex subunit 7

CCR4-NOT transcription complex subunit 7/8/Pop2 Gene3D Ribonuclease H superfamily

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 285

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

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