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Mouse Rec8 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rec8 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rec8 gene (NCBI Reference Sequence: NM_020002 ; Ensembl: ENSMUSG00000002324 ) is located on Mouse chromosome 14. 20 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 20 (Transcript: ENSMUST00000002395). Exon 7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rec8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-351I22 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 are infertile and exhibit small ovaries and testes. Females show absence of ovarian follicles and abnormal meiosis, while males exhibit abnormal chromosome pairing during meiosis, abnormal synaptonemal complex formation, and arrest of male meiosis.

Exon 7 starts from about 26.28% of the coding region. The knockout of Exon 7 will result in frameshift of the gene. The size of intron 6 for 5'-loxP site insertion: 1534 bp, and the size of intron 7 for 3'-loxP site insertion: 895 bp. The size of effective cKO region: ~585 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 6 7 8 9 10 11 12 13 20 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rec8 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(7085bp) | A(25.22% 1787) | C(25.69% 1820) | T(23.23% 1646) | G(25.86% 1832)

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% chr14 + 55617986 55620985 3000 browser details YourSeq 111 2719 2917 3000 91.1% chr12 + 25006066 25006654 589 browser details YourSeq 95 2716 2863 3000 92.0% chr10 + 91050041 91050226 186 browser details YourSeq 93 2632 2799 3000 81.8% chr2 - 25393758 25393901 144 browser details YourSeq 88 2625 2799 3000 88.6% chr19 - 47793431 47793715 285 browser details YourSeq 88 2716 2861 3000 88.4% chr11 - 109955287 109955481 195 browser details YourSeq 88 2718 2837 3000 89.2% chr19 + 32098684 32098826 143 browser details YourSeq 84 1263 1572 3000 95.8% chr10 + 59432122 59432507 386 browser details YourSeq 83 2716 2862 3000 87.4% chr13 + 98380206 98380391 186 browser details YourSeq 82 2715 2860 3000 87.3% chr1 - 161067656 161067837 182 browser details YourSeq 79 2690 2799 3000 82.3% chr7 - 50500787 50500882 96 browser details YourSeq 75 2714 2796 3000 95.2% chr1 - 121513526 121513608 83 browser details YourSeq 74 2703 2799 3000 83.6% chr6 + 35953224 35953314 91 browser details YourSeq 73 2716 2802 3000 92.0% chr11 + 33451476 33451562 87 browser details YourSeq 72 2716 2799 3000 92.9% chr16 - 87775008 87775091 84 browser details YourSeq 72 2716 2799 3000 90.4% chr8 + 111698961 111699043 83 browser details YourSeq 72 2716 2799 3000 92.9% chr14 + 8045976 8046059 84 browser details YourSeq 72 2716 2799 3000 92.9% chr10 + 76109286 76109369 84 browser details YourSeq 72 2713 2806 3000 87.0% chr1 + 10114759 10114851 93 browser details YourSeq 71 2716 2802 3000 90.9% chr13 - 106917215 106917301 87

Note: The 3000 bp section upstream of Exon 7 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% chr14 + 55621571 55624570 3000 browser details YourSeq 268 1 329 3000 92.0% chr5 + 110626290 110626961 672 browser details YourSeq 250 12 333 3000 87.4% chr17 + 47393611 47393928 318 browser details YourSeq 250 12 333 3000 92.5% chr13 + 111740100 111740537 438 browser details YourSeq 247 4 324 3000 89.8% chr17 - 26271986 26272630 645 browser details YourSeq 247 4 329 3000 90.5% chr7 + 6166699 6167194 496 browser details YourSeq 242 4 320 3000 91.2% chr11 - 117701222 117915431 214210 browser details YourSeq 242 4 333 3000 90.4% chr16 + 13873578 13874263 686 browser details YourSeq 237 4 527 3000 89.3% chr14 - 64971472 64972073 602 browser details YourSeq 235 13 329 3000 91.1% chr13 + 37736778 38049580 312803 browser details YourSeq 227 1 333 3000 92.3% chr8 + 71866593 71866926 334 browser details YourSeq 226 4 310 3000 87.7% chr19 - 38399864 38400220 357 browser details YourSeq 206 4 285 3000 88.7% chr7 - 44807651 44808047 397 browser details YourSeq 198 35 528 3000 91.3% chr11 - 70901149 70901704 556 browser details YourSeq 187 1 326 3000 87.8% chr18 - 34977215 34977671 457 browser details YourSeq 182 26 324 3000 92.6% chr7 + 141377786 141378307 522 browser details YourSeq 181 6 313 3000 87.9% chr2 + 153158873 153159181 309 browser details YourSeq 179 1 527 3000 82.4% chr19 + 36895134 36895461 328 browser details YourSeq 178 11 300 3000 91.3% chr16 + 32621964 32622546 583 browser details YourSeq 170 4 310 3000 86.1% chr4 + 136091022 136091326 305

Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rec8 REC8 meiotic recombination protein [ Mus musculus (house mouse) ] Gene ID: 56739, updated on 10-Oct-2019

Gene summary

Official Symbol Rec8 provided by MGI Official Full Name REC8 meiotic recombination protein provided by MGI Primary source MGI:MGI:1929645 See related Ensembl:ENSMUSG00000002324 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 mrec; Rec8L1 Expression Broad expression in testis adult (RPKM 25.9), duodenum adult (RPKM 18.4) and 17 other tissues See more Orthologs human all

Genomic context

Location: 14 C3; 14 28.19 cM See Rec8 in Genome Data Viewer

Exon count: 20

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (55618002..55625395)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (56237007..56244227)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 4 transcripts

Gene: Rec8 ENSMUSG00000002324

Description REC8 meiotic recombination protein [Source:MGI Symbol;Acc:MGI:1929645] Gene Synonyms Rec8L1, mrec Location Chromosome 14: 55,618,037-55,625,395 forward strand. GRCm38:CM001007.2 About this gene This gene has 4 transcripts (splice variants), 151 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 23 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rec8-201 ENSMUST00000002395.7 2184 591aa ENSMUSP00000002395.7 Protein coding CCDS27120 Q8C5S7 TSL:1 GENCODE basic APPRIS P1

Rec8-202 ENSMUST00000141425.1 836 No protein - Retained intron - - TSL:3

Rec8-204 ENSMUST00000227922.1 726 No protein - Retained intron - - -

Rec8-203 ENSMUST00000155193.1 381 No protein - Retained intron - - TSL:2

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27.36 kb Forward strand 55.61Mb 55.62Mb 55.63Mb Genes (Comprehensive set... Irf9-203 >protein coding Rec8-201 >protein coding

Irf9-202 >protein coding Rec8-204 >retained intron

Irf9-205 >protein coding Rec8-202 >retained intron

Irf9-206 >retained intron Rec8-203 >retained intron

Irf9-201 >retained intron

Irf9-207 >protein coding

Contigs < AC174678.2

Genes < Ipo4-201protein coding (Comprehensive set...

< Ipo4-209nonsense mediated decay

< Ipo4-207retained intron < Ipo4-211retained intron

< Ipo4-212lncRNA < Ipo4-204retained intron

< Ipo4-208lncRNA < Gm49378-201nonsense mediated decay

< Ipo4-206protein coding < Ipo4-210retained intron

< Ipo4-205nonsense mediated decay

< Ipo4-203retained intron < Ipo4-202protein coding

< Ipo4-213lncRNA

Regulatory Build

55.61Mb 55.62Mb 55.63Mb Reverse strand 27.36 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000002395

7.36 kb Forward strand

Rec8-201 >protein coding

ENSMUSP00000002... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Winged helix DNA-binding domain superfamily Pfam Rad21/Rec8-like protein, N-terminal Rad21/Rec8-like protein, C-terminal, eukaryotic

PANTHER Rad21/Rec8-like protein

PTHR12585:SF27

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 591

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

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