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Mouse Tarbp2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tarbp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tarbp2 gene (NCBI Reference Sequence: NM_009319 ; Ensembl: ENSMUSG00000023051 ) is located on Mouse chromosome 15. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000023813). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tarbp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-301N23 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 inactivation of this gene results in lethality at weaning, decreased body weight, and male infertility associated with oligozoospermia and failure of spermiation.

Exon 3 starts from about 20.46% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1183 bp, and the size of intron 4 for 3'-loxP site insertion: 510 bp. The size of effective cKO region: ~1239 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 4 5 6 7 8 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Tarbp2 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(7739bp) | A(22.15% 1714) | C(26.67% 2064) | T(23.85% 1846) | G(27.33% 2115)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr15 + 102517226 102520225 3000 browser details YourSeq 227 1564 2068 3000 92.2% chr6 - 47432920 47433147 228 browser details YourSeq 110 1230 1379 3000 93.7% chr7 + 127154953 127155123 171 browser details YourSeq 52 2790 2904 3000 92.0% chr1 + 87563023 87563140 118 browser details YourSeq 51 2784 2901 3000 85.0% chr9 + 14954580 14954698 119 browser details YourSeq 48 2872 2947 3000 85.3% chr3 - 67671490 67671566 77 browser details YourSeq 43 2767 2826 3000 86.7% chrX - 38815614 38815680 67 browser details YourSeq 41 2866 2914 3000 91.9% chr13 + 20436490 20436538 49 browser details YourSeq 39 2809 2903 3000 70.6% chr19 - 29999387 29999481 95 browser details YourSeq 39 2627 2904 3000 93.4% chr11 - 114497602 114498041 440 browser details YourSeq 38 2617 2826 3000 88.0% chr2 - 180280274 180280483 210 browser details YourSeq 38 2872 2926 3000 86.6% chr2 + 132969811 132969866 56 browser details YourSeq 37 2885 2926 3000 95.3% chr9 - 91918636 91918678 43 browser details YourSeq 36 2886 2928 3000 97.4% chr1 - 119277808 119277851 44 browser details YourSeq 36 2872 2909 3000 97.4% chr6 + 93654888 93654925 38 browser details YourSeq 36 2875 2925 3000 92.9% chr1 + 52321226 52321277 52 browser details YourSeq 33 2783 2819 3000 94.6% chr2 + 29356003 29356039 37 browser details YourSeq 32 2776 2809 3000 97.1% chr9 - 60699350 60699383 34 browser details YourSeq 32 2873 2919 3000 90.0% chr2 - 53766643 53766694 52 browser details YourSeq 32 2789 2826 3000 92.2% chr1 - 75128111 75128148 38

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% chr15 + 102521465 102524464 3000 browser details YourSeq 931 515 2212 3000 97.2% chr6 - 47431786 47432727 942 browser details YourSeq 853 515 2191 3000 93.2% chr7 + 127155316 127156262 947 browser details YourSeq 33 851 958 3000 94.6% chr3 + 89491347 89491458 112 browser details YourSeq 31 762 796 3000 97.0% chr4 + 135956346 135956433 88 browser details YourSeq 25 2592 2617 3000 100.0% chr3 - 88535765 88535791 27 browser details YourSeq 25 1731 1760 3000 96.5% chr1 - 5959685 5959714 30 browser details YourSeq 25 735 762 3000 84.7% chr1 + 175644741 175644766 26 browser details YourSeq 24 1662 1685 3000 100.0% chr11 - 45504904 45504927 24 browser details YourSeq 23 2557 2579 3000 100.0% chr6 + 57466461 57466483 23 browser details YourSeq 21 627 647 3000 100.0% chr17 + 90502571 90502591 21

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: Tarbp2 TARBP2, RISC loading complex RNA binding subunit [ Mus musculus (house mouse) ] Gene ID: 21357, updated on 12-Aug-2019

Gene summary

Official Symbol Tarbp2 provided by MGI Official Full Name TARBP2, RISC loading complex RNA binding subunit provided by MGI Primary source MGI:MGI:103027 See related Ensembl:ENSMUSG00000023051 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 Prbp; TRBP Expression Ubiquitous expression in testis adult (RPKM 86.4), thymus adult (RPKM 26.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 15 F3; 15 57.65 cM See Tarbp2 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (102516077..102523676)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (102348677..102354107)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 14 transcripts

Gene: Tarbp2 ENSMUSG00000023051

Description TARBP2, RISC loading complex RNA binding subunit [Source:MGI Symbol;Acc:MGI:103027] Gene Synonyms Prbp, TRBP Location Chromosome 15: 102,518,192-102,523,676 forward strand. GRCm38:CM001008.2 About this gene This gene has 14 transcripts (splice variants), 203 orthologues, 14 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tarbp2- ENSMUST00000023813.8 1523 365aa ENSMUSP00000023813.2 Protein coding CCDS27886 P97473 TSL:1 201 GENCODE basic APPRIS P1

Tarbp2- ENSMUST00000100168.9 1278 274aa ENSMUSP00000097744.3 Protein coding CCDS57013 Q3UNH9 TSL:1 202 GENCODE basic

Tarbp2- ENSMUST00000146756.7 1081 225aa ENSMUSP00000121748.1 Protein coding - D3Z282 CDS 3' 208 incomplete TSL:3

Tarbp2- ENSMUST00000142194.2 782 243aa ENSMUSP00000123339.1 Protein coding - D3YZS5 CDS 3' 207 incomplete TSL:3

Tarbp2- ENSMUST00000150393.8 489 89aa ENSMUSP00000120315.2 Protein coding - D3Z033 CDS 3' 210 incomplete TSL:5

Tarbp2- ENSMUST00000229805.1 377 42aa ENSMUSP00000155826.1 Protein coding - A0A2R8VI89 CDS 3' 214 incomplete

Tarbp2- ENSMUST00000131184.7 1819 79aa ENSMUSP00000117964.1 Nonsense mediated - D6RE49 TSL:1 203 decay

Tarbp2- ENSMUST00000154948.7 951 111aa ENSMUSP00000154902.1 Nonsense mediated - A0A2R8VHA7 CDS 5' 213 decay incomplete TSL:3

Tarbp2- ENSMUST00000149200.7 482 58aa ENSMUSP00000123213.1 Nonsense mediated - D6RCY4 TSL:3 209 decay

Tarbp2- ENSMUST00000136968.1 2732 No - Retained intron - - TSL:2 205 protein

Tarbp2- ENSMUST00000141266.7 1033 No - Retained intron - - TSL:2 206 protein

Tarbp2- ENSMUST00000134033.7 713 No - Retained intron - - TSL:2 204 protein

Tarbp2- ENSMUST00000153171.1 707 No - Retained intron - - TSL:2 211 protein

Tarbp2- ENSMUST00000154451.1 704 No - Retained intron - - TSL:2 212 protein

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25.48 kb Forward strand 102.51Mb 102.52Mb 102.53Mb Genes (Comprehensive set... Tarbp2-203 >nonsense mediated decay

Tarbp2-206 >retained intron

Tarbp2-202 >protein coding

Tarbp2-205 >retained intron

Tarbp2-214 >protein coding

Tarbp2-210 >protein coding

Tarbp2-201 >protein coding

Tarbp2-212 >retained intron

Tarbp2-213 >nonsense mediated decay

Tarbp2-204 >retained intron

Tarbp2-208 >protein coding

Tarbp2-209 >nonsense mediated decay

Tarbp2-207 >protein coding

Tarbp2-211 >retained intron

Contigs AC137156.3 >

Genes < Map3k12-201protein coding < Npff-201protein coding < Atf7-210protein coding (Comprehensive set...

< Map3k12-208protein coding < Gm28047-201nonsense mediated decay

< Map3k12-207protein coding < Npff-202retained intron < Atf7-201protein coding

< Map3k12-206protein coding < Atf7-211protein coding

< Map3k12-203protein coding < Atf7-209protein coding

< Map3k12-205protein coding

< Map3k12-202retained intron

< Map3k12-204retained intron

Regulatory Build

102.51Mb 102.52Mb 102.53Mb Reverse strand 25.48 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000023813

5.22 kb Forward strand

Tarbp2-201 >protein coding

ENSMUSP00000023... Low complexity (Seg) Superfamily SSF54768 SMART Double-stranded RNA-binding domain Pfam Double-stranded RNA-binding domain PROSITE profiles Double-stranded RNA-binding domain PANTHER PTHR46205

RISC-loading complex subunit TRBP2 HAMAP RISC-loading complex subunit TRBP2 Gene3D 3.30.160.20 CDD Double-stranded RNA-binding domain

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 365

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

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