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Mouse Uqcr11 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Uqcr11 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Uqcr11 gene (NCBI Reference Sequence: NM_025650 ; Ensembl: ENSMUSG00000020163 ) is located on Mouse chromosome 10. 3 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000020372). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Uqcr11 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-132N8 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:

Exon 2 starts from about 30.36% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2409 bp, and the size of intron 2 for 3'-loxP site insertion: 980 bp. The size of effective cKO region: ~618 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Uqcr11 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(7118bp) | A(23.48% 1671) | C(25.26% 1798) | T(25.5% 1815) | G(25.77% 1834)

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% chr10 - 80404537 80407536 3000 browser details YourSeq 95 742 842 3000 97.1% chr11 - 102492559 102492659 101 browser details YourSeq 90 2262 2972 3000 97.0% chr3 + 104574355 104687771 113417 browser details YourSeq 85 739 842 3000 91.3% chr11 + 52405259 52405365 107 browser details YourSeq 71 772 842 3000 100.0% chr1 - 166431825 166431895 71 browser details YourSeq 59 1255 1370 3000 77.8% chr2 - 163853065 163853179 115 browser details YourSeq 53 2930 2988 3000 96.6% chr19 + 28711235 28711324 90 browser details YourSeq 52 2122 2292 3000 88.1% chr11 - 77201215 77201715 501 browser details YourSeq 51 2934 3000 3000 93.4% chr1 - 15640058 15640152 95 browser details YourSeq 46 2238 2333 3000 87.1% chr11 - 100834558 100834670 113 browser details YourSeq 44 2933 2980 3000 98.0% chr7 - 15960282 15960331 50 browser details YourSeq 44 2930 2981 3000 94.2% chr10 - 44317932 44317985 54 browser details YourSeq 43 2239 2293 3000 89.1% chr16 - 25382599 25382653 55 browser details YourSeq 43 2242 2335 3000 89.1% chr8 + 95851219 95851335 117 browser details YourSeq 43 2947 3000 3000 94.0% chr10 + 5886474 5886554 81 browser details YourSeq 42 2934 2981 3000 95.8% chr11 - 47656823 47656872 50 browser details YourSeq 42 2931 2980 3000 93.9% chr13 + 38685355 38685406 52 browser details YourSeq 41 2237 2334 3000 90.2% chr12 + 109702490 109702593 104 browser details YourSeq 41 2942 2988 3000 95.8% chr1 + 127974360 127974424 65 browser details YourSeq 40 2934 2981 3000 93.7% chr10 - 80203238 80203287 50

Note: The 3000 bp section upstream of Exon 2 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% chr10 - 80400919 80403918 3000 browser details YourSeq 229 2201 2519 3000 91.7% chr10 - 121319708 121320480 773 browser details YourSeq 227 2201 2517 3000 87.7% chr12 - 111399645 111399962 318 browser details YourSeq 225 2201 2517 3000 88.2% chr10 - 63180567 63180884 318 browser details YourSeq 225 2205 2517 3000 87.9% chr14 + 65160237 65160550 314 browser details YourSeq 224 2200 2519 3000 89.6% chr10 - 9237736 9238060 325 browser details YourSeq 223 2201 2510 3000 88.9% chr5 + 92984283 92984592 310 browser details YourSeq 221 2201 2512 3000 87.2% chr13 - 46037730 46038042 313 browser details YourSeq 221 2202 2517 3000 89.9% chr13 + 101828777 101829097 321 browser details YourSeq 220 2201 2517 3000 87.0% chr5 - 103217436 103217752 317 browser details YourSeq 220 2196 2517 3000 88.6% chr17 - 29540902 29541227 326 browser details YourSeq 218 2212 2518 3000 89.5% chr9 + 45214736 45215065 330 browser details YourSeq 217 2212 2522 3000 88.1% chr9 - 97189308 97189620 313 browser details YourSeq 217 2204 2518 3000 88.2% chr10 - 105856157 105856472 316 browser details YourSeq 217 2201 2512 3000 90.2% chr1 - 157091528 157109185 17658 browser details YourSeq 215 2212 2518 3000 89.1% chr7 - 82210930 82211238 309 browser details YourSeq 215 2201 2518 3000 90.7% chr1 - 152748125 152748445 321 browser details YourSeq 215 2201 2525 3000 86.7% chr1 + 161738337 161738662 326 browser details YourSeq 215 2204 2512 3000 88.3% chr1 + 128530720 128531260 541 browser details YourSeq 214 2203 2519 3000 87.7% chr7 - 139142554 139142873 320

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Uqcr11 ubiquinol-cytochrome c reductase, complex III subunit XI [ Mus musculus (house mouse) ] Gene ID: 66594, updated on 12-Aug-2019

Gene summary

Official Symbol Uqcr11 provided by MGI Official Full Name ubiquinol-cytochrome c reductase, complex III subunit XI provided by MGI Primary source MGI:MGI:1913844 See related Ensembl:ENSMUSG00000020163 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 Uqcr; AL022707; 0710008D09Rik Expression Ubiquitous expression in heart adult (RPKM 1055.3), kidney adult (RPKM 899.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 C1 See Uqcr11 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (80402997..80406821, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (79865742..79869566, complement)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 3 transcripts

Gene: Uqcr11 ENSMUSG00000020163

Description ubiquinol-cytochrome c reductase, complex III subunit XI [Source:MGI Symbol;Acc:MGI:1913844] Gene Synonyms 0710008D09Rik, Uqcr Location Chromosome 10: 80,402,997-80,406,830 reverse strand. GRCm38:CM001003.2 About this gene This gene has 3 transcripts (splice variants), 150 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Uqcr11-201 ENSMUST00000020372.5 445 56aa ENSMUSP00000020372.5 Protein coding CCDS35981 Q9CPX8 TSL:1 GENCODE basic APPRIS P1

Uqcr11-203 ENSMUST00000141683.1 605 No protein - lncRNA - - TSL:1

Uqcr11-202 ENSMUST00000138428.7 520 No protein - lncRNA - - TSL:3

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23.83 kb Forward strand 80.395Mb 80.400Mb 80.405Mb 80.410Mb 80.415Mb Contigs < AC152062.6 Genes (Comprehensive set... < Mbd3-204protein coding < Uqcr11-201protein coding < Tcf3-209protein coding

< Mbd3-201protein coding < Uqcr11-202lncRNA < Tcf3-208protein coding

< Mbd3-206protein coding < Uqcr11-203lncRNA < Tcf3-207protein coding

< Mbd3-203protein coding < Tcf3-206protein coding

< Mbd3-202protein coding < Tcf3-201protein coding

< Mbd3-205lncRNA < Tcf3-210protein coding

< Tcf3-205protein coding

< Tcf3-202protein coding

< Tcf3-204protein coding

< Tcf3-203protein coding

< Tcf3-214protein coding

< Tcf3-212lncRNA

< Tcf3-211retained intron

Regulatory Build

80.395Mb 80.400Mb 80.405Mb 80.410Mb 80.415Mb Reverse strand 23.83 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000020372

< Uqcr11-201protein coding

Reverse strand 3.83 kb

ENSMUSP00000020... Transmembrane heli... Superfamily Single alpha-helix domain superfamily Pfam Cytochrome b-c1 complex subunit 10 PANTHER Cytochrome b-c1 complex subunit 10 Gene3D 1.20.5.220

Scale bar 0 6 12 18 24 30 36 42 48 56

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

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