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Mouse Prpf3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Prpf3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Prpf3 (NCBI Reference Sequence: NM_027541 ; Ensembl: ENSMUSG00000015748 ) is located on Mouse 3. 16 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000161476). 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 Prpf3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-456L24 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: Mice homozygous for a null mutation display embryonic lethality.

Exon 3 starts from about 7.13% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of 2 for 5'-loxP site insertion: 1750 bp, and the size of intron 3 for 3'-loxP site insertion: 2501 bp. The size of effective cKO region: ~631 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 2 3 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Prpf3 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(7131bp) | A(28.0% 1997) | C(18.82% 1342) | T(30.8% 2196) | G(22.38% 1596)

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% chr3 - 95851944 95854943 3000 browser details YourSeq 123 2784 2980 3000 91.9% chr7 + 132945432 132945871 440 browser details YourSeq 121 2778 2942 3000 87.8% chr13 - 82437339 82437513 175 browser details YourSeq 119 2790 2950 3000 85.3% chr12 - 75685555 75685704 150 browser details YourSeq 116 2786 2954 3000 82.0% chr7 - 19989432 19989591 160 browser details YourSeq 113 1849 2037 3000 84.9% chr12 + 84314214 84314404 191 browser details YourSeq 109 1305 1441 3000 89.8% chr4 - 8673569 8673705 137 browser details YourSeq 108 2784 2912 3000 92.3% chr8 + 89334610 89334740 131 browser details YourSeq 105 2786 2916 3000 90.1% chr17 + 46925385 46925515 131 browser details YourSeq 104 2786 2913 3000 90.7% chr7 - 64170769 64170896 128 browser details YourSeq 104 2790 2929 3000 89.4% chr3 - 96753666 96753807 142 browser details YourSeq 102 2786 2913 3000 89.9% chr2 - 149098876 149099003 128 browser details YourSeq 102 2794 2916 3000 89.0% chr6 + 87051502 87051620 119 browser details YourSeq 101 1876 2037 3000 85.0% chr18 - 78265548 78265712 165 browser details YourSeq 98 1833 2037 3000 78.8% chr6 + 24639124 24639320 197 browser details YourSeq 92 1884 2046 3000 83.4% chr18 - 74735074 74735239 166 browser details YourSeq 88 1856 2037 3000 77.9% chr9 + 63847944 63848096 153 browser details YourSeq 86 1884 2037 3000 75.2% chr1 - 57877802 57877951 150 browser details YourSeq 85 1885 2035 3000 79.8% chr11 + 52560720 52560872 153 browser details YourSeq 82 1884 2041 3000 84.2% chr1 + 136708575 136708732 158

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% chr3 - 95848313 95851312 3000 browser details YourSeq 108 707 3000 3000 89.3% chr5 - 142923132 143117881 194750 browser details YourSeq 76 671 775 3000 90.6% chr13 - 107832601 107832735 135 browser details YourSeq 71 724 862 3000 83.9% chr6 - 55000195 55000332 138 browser details YourSeq 69 2814 3000 3000 90.7% chr13 + 44773468 44773854 387 browser details YourSeq 67 707 2906 3000 85.3% chr5 - 129985903 130008537 22635 browser details YourSeq 67 709 853 3000 83.9% chr2 - 70497293 70497440 148 browser details YourSeq 67 668 851 3000 79.6% chr13 + 79312017 79312191 175 browser details YourSeq 63 709 869 3000 87.9% chr16 + 30813821 30813981 161 browser details YourSeq 59 2813 2890 3000 88.5% chr13 + 15196359 15196437 79 browser details YourSeq 58 677 794 3000 80.7% chr11 - 102412765 102412881 117 browser details YourSeq 58 2813 2891 3000 87.4% chr2 + 117131748 117131828 81 browser details YourSeq 58 2813 2892 3000 87.5% chr12 + 76042947 76043031 85 browser details YourSeq 57 2812 2890 3000 91.4% chr1 - 80406255 80406334 80 browser details YourSeq 56 2817 2889 3000 89.1% chr5 - 20029934 20030008 75 browser details YourSeq 55 2828 3000 3000 91.1% chr2 - 132755915 132756356 442 browser details YourSeq 54 2813 2889 3000 88.6% chr1 - 82280646 82280726 81 browser details YourSeq 53 2847 3000 3000 92.1% chr3 - 9637788 9637954 167 browser details YourSeq 52 2852 3000 3000 94.9% chr13 - 96444726 96445263 538 browser details YourSeq 51 719 795 3000 83.2% chr11 - 86509132 86509208 77

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 information: Prpf3 pre-mRNA processing factor 3 [ Mus musculus (house mouse) ] Gene ID: 70767, updated on 15-Aug-2019

Gene summary

Official Symbol Prpf3 provided by MGI Official Full Name pre-mRNA processing factor 3 provided by MGI Primary source MGI:MGI:1918017 See related Ensembl:ENSMUSG00000015748 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 3632413F13Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 11.3), liver E14 (RPKM 7.8) and 24 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 F2.1 See Prpf3 in Genome Data Viewer

Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (95830124..95855885, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (95634545..95659676, complement)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 9 transcripts

Gene: Prpf3 ENSMUSG00000015748

Description pre-mRNA processing factor 3 [Source:MGI Symbol;Acc:MGI:1918017] Gene Synonyms 3632413F13Rik Location Chromosome 3: 95,830,124-95,855,885 reverse strand. GRCm38:CM000996.2 About this gene This gene has 9 transcripts (splice variants), 200 orthologues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Prpf3- ENSMUST00000015892.13 2933 683aa ENSMUSP00000015892.7 Protein coding CCDS17621 Q922U1 TSL:1 201 GENCODE basic APPRIS P1

Prpf3- ENSMUST00000161476.7 2732 683aa ENSMUSP00000124950.1 Protein coding CCDS17621 Q922U1 TSL:1 207 GENCODE basic APPRIS P1

Prpf3- ENSMUST00000159901.7 575 114aa ENSMUSP00000124444.1 Protein coding - F6T4V0 CDS 5' 202 incomplete TSL:3

Prpf3- ENSMUST00000160109.1 800 101aa ENSMUSP00000124302.1 Nonsense mediated - E0CYD0 TSL:3 203 decay

Prpf3- ENSMUST00000160155.7 2548 No - Retained intron - - TSL:1 204 protein

Prpf3- ENSMUST00000161420.1 1800 No - Retained intron - - TSL:1 206 protein

Prpf3- ENSMUST00000161073.1 613 No - Retained intron - - TSL:3 205 protein

Prpf3- ENSMUST00000163059.1 592 No - Retained intron - - TSL:2 209 protein

Prpf3- ENSMUST00000161482.1 554 No - lncRNA - - TSL:3 208 protein

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45.76 kb Forward strand 95.83Mb 95.84Mb 95.85Mb 95.86Mb Gm26594-201 >lncRNA (Comprehensive set...

Contigs < AC093317.22

Genes (Comprehensive set... < Prpf3-201protein coding < Mrps21-201protein coding

< Prpf3-202protein coding < Prpf3-206retained intron < Mrps21-202protein coding

< Prpf3-204retained intron < Mrps21-204protein coding

< Prpf3-207protein coding < Mrps21-203lncRNA

< Prpf3-209retained intron < Prpf3-203nonsense mediated decay

< Prpf3-208lncRNA < Prpf3-205retained intron

Regulatory Build

95.83Mb 95.84Mb 95.85Mb 95.86Mb Reverse strand 45.76 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

RNA gene processed transcript

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Transcript: ENSMUST00000161476

< Prpf3-207protein coding

Reverse strand 25.13 kb

ENSMUSP00000124... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily PWI domain superfamily SMART PWI domain Pfam PWI domain Pre-mRNA-splicing factor 3 Domain of unknown function DUF1115

PROSITE profiles PWI domain PANTHER U4/U6 small nuclear ribonucleoprotein Prp3 Gene3D 1.20.1390.10

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 683

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

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