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Mouse Tex35 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tex35 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tex35 gene (NCBI Reference Sequence: NM_028540 ; Ensembl: ENSMUSG00000026592 ) is located on Mouse chromosome 1. 9 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000118207). Exon 6~9 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tex35 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-90N4 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: Male mice homozygous for a null allele exhibit normal fecundity.

Exon 6~9 covers 56.73% of the coding region. Start codon is in exon 1, and stop codon is in exon 9. The size of intron 5 for 5'-loxP site insertion: 3819 bp. The size of effective cKO region: ~2186 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 6 7 8 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tex35 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(7679bp) | A(26.58% 2041) | C(23.56% 1809) | T(27.5% 2112) | G(22.36% 1717)

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 - 157100775 157103774 3000 browser details YourSeq 64 1222 1328 3000 94.6% chr4 + 138665146 138665423 278 browser details YourSeq 59 1171 1258 3000 81.4% chr4 - 45541250 45541336 87 browser details YourSeq 54 1196 1314 3000 85.9% chr14 - 80053964 80054091 128 browser details YourSeq 52 1170 1259 3000 78.9% chr5 + 119762211 119762300 90 browser details YourSeq 52 2839 2907 3000 94.7% chr11 + 56572383 56572451 69 browser details YourSeq 50 1222 1326 3000 93.3% chr1 - 54732059 54732561 503 browser details YourSeq 48 1198 1261 3000 87.5% chr1 - 93105216 93105279 64 browser details YourSeq 43 1222 1328 3000 75.6% chr12 + 84610505 84610594 90 browser details YourSeq 43 1206 1317 3000 74.6% chr11 + 103353407 103353503 97 browser details YourSeq 39 1196 1254 3000 83.1% chr6 - 29704945 29705003 59 browser details YourSeq 37 1170 1261 3000 84.5% chr1 + 86914817 86914906 90 browser details YourSeq 36 1222 1261 3000 95.0% chr4 - 57925658 57925697 40 browser details YourSeq 36 1202 1261 3000 87.8% chr12 - 111984715 111984776 62 browser details YourSeq 36 1207 1261 3000 87.8% chr12 - 108582316 108582375 60 browser details YourSeq 36 1222 1261 3000 95.0% chr10 + 17665051 17665090 40 browser details YourSeq 35 1222 1261 3000 95.0% chr5 - 135799956 135799996 41 browser details YourSeq 35 1219 1261 3000 91.9% chr4 - 57021483 57021524 42 browser details YourSeq 35 1205 1260 3000 72.0% chr13 - 103939883 103939932 50 browser details YourSeq 35 1206 1261 3000 80.9% chr1 - 84951031 84951084 54

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% chr1 - 157096346 157099345 3000 browser details YourSeq 1035 1914 3000 3000 98.1% chr9 + 16704933 16706151 1219 browser details YourSeq 971 1905 3000 3000 97.6% chrX - 44611653 44612778 1126 browser details YourSeq 970 1697 2753 3000 96.6% chrX - 146850622 146851681 1060 browser details YourSeq 852 1905 2796 3000 98.4% chr9 + 33008309 33009646 1338 browser details YourSeq 821 1906 2753 3000 99.0% chrX + 104835452 104836301 850 browser details YourSeq 821 1880 2753 3000 98.0% chr11 + 39436940 39437819 880 browser details YourSeq 820 1909 2753 3000 99.1% chr9 - 54928275 54929121 847 browser details YourSeq 819 1745 2753 3000 97.7% chrX - 77321267 77322450 1184 browser details YourSeq 817 1906 2753 3000 98.7% chr10 + 57626117 57626966 850 browser details YourSeq 816 1906 2753 3000 98.7% chr4 + 74958132 74958984 853 browser details YourSeq 815 1907 2753 3000 98.7% chr18 - 80916137 80917164 1028 browser details YourSeq 815 1906 2753 3000 98.6% chr2 + 113671827 113672676 850 browser details YourSeq 814 1907 2753 3000 98.6% chr17 - 45202037 45202885 849 browser details YourSeq 813 1906 2753 3000 98.5% chr3 + 119259604 119260453 850 browser details YourSeq 812 1906 2753 3000 98.3% chr6 + 138741471 138742325 855 browser details YourSeq 812 1906 2753 3000 98.5% chr1 + 93894094 93894945 852 browser details YourSeq 811 1906 2753 3000 98.4% chr5 - 91837502 91838351 850 browser details YourSeq 811 1906 2753 3000 98.4% chr5 - 78358357 78359206 850 browser details YourSeq 811 1908 2753 3000 98.5% chr4 - 104627632 104628479 848

Note: The 3000 bp section downstream of Exon 9 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tex35 testis expressed 35 [ Mus musculus (house mouse) ] Gene ID: 73435, updated on 24-Oct-2019

Gene summary

Official Symbol Tex35 provided by MGI Official Full Name testis expressed 35 provided by MGI Primary source MGI:MGI:1920685 See related Ensembl:ENSMUSG00000026592 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 1700057K13Rik Expression Restricted expression toward testis adult (RPKM 77.3) See more Orthologs human all

Genomic context

Location: 1; 1 G3 See Tex35 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (157099141..157108680, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (159029279..159038776, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 6 transcripts

Gene: Tex35 ENSMUSG00000026592

Description testis expressed 35 [Source:MGI Symbol;Acc:MGI:1920685] Gene Synonyms 1700057K13Rik Location Chromosome 1: 157,099,139-157,108,680 reverse strand. GRCm38:CM000994.2 About this gene This gene has 6 transcripts (splice variants), 82 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tex35- ENSMUST00000118207.7 912 208aa ENSMUSP00000114092.1 Protein coding CCDS48407 A0A0R4J1P6 TSL:1 202 GENCODE basic APPRIS P3

Tex35- ENSMUST00000027884.12 909 207aa ENSMUSP00000027884.6 Protein coding CCDS78718 A0A0R4J070 TSL:1 201 GENCODE basic APPRIS ALT2

Tex35- ENSMUST00000121911.7 805 187aa ENSMUSP00000113622.1 Protein coding CCDS78716 D3Z4U7 TSL:5 204 GENCODE basic APPRIS ALT2

Tex35- ENSMUST00000119526.1 679 186aa ENSMUSP00000113325.1 Protein coding CCDS78715 D3Z652 TSL:5 203 GENCODE basic APPRIS ALT2

Tex35- ENSMUST00000187546.6 856 209aa ENSMUSP00000139460.1 Protein coding - A0A087WNR4 TSL:5 206 GENCODE basic APPRIS ALT2

Tex35- ENSMUST00000148649.2 820 55aa ENSMUSP00000116924.1 Nonsense mediated - D6RGU0 TSL:5 205 decay

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29.54 kb Forward strand

157.09Mb 157.10Mb 157.11Mb Contigs < AC119220.11

Genes (Comprehensive set... < Tex35-201protein coding

< Tex35-202protein coding

< Tex35-206protein coding

< Tex35-204protein coding

< Tex35-203protein coding

< Tex35-205nonsense mediated decay

Regulatory Build

157.09Mb 157.10Mb 157.11Mb Reverse strand 29.54 kb

Regulation Legend CTCF Enhancer Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000118207

< Tex35-202protein coding

Reverse strand 9.51 kb

ENSMUSP00000114... Coiled-coils (Ncoils) Pfam Testis-expressed sequence 35 protein PANTHER Testis-expressed sequence 35 protein

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 208

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

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