Mouse Rsl1d1 Conditional Knockout Project (CRISPR/Cas9)

http://www.alphaknockout.com/ Mouse Rsl1d1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rsl1d1 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rsl1d1 gene ( NCBI Reference Sequence: NM_025546 ; Ensembl: ENSMUSG00000005846 ) is located on mouse chromosome 16. 9 exons are identified , with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000119953). Exon 2~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Rsl1d1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-325B2 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:

The knockout of Exon 2~5 will result in frameshift of the gene, and covers 38.86% of the coding region. The size of intron 1 for 5'-loxP site insertion: 693 bp, and the size of intron 5 for 3'-loxP site insertion: 2818 bp. The size of effective cKO region: ~3572 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 5 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Rsl1d1 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(10073bp) | A(26.28% 2647) | C(21.27% 2143) | G(22.92% 2309) | T(29.52% 2974)

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% chr16 - 11202708 11205707 3000 browser details YourSeq 96 989 1111 3000 89.8% chr10 - 113048057 113048174 118 browser details YourSeq 95 798 1095 3000 91.2% chr1 - 34585972 34586272 301 browser details YourSeq 95 989 1112 3000 89.2% chr15 + 8456381 8456505 125 browser details YourSeq 94 989 1112 3000 87.9% chr1 - 72774077 72774194 118 browser details YourSeq 94 989 1112 3000 90.3% chr9 + 65817838 65817960 123 browser details YourSeq 93 798 1112 3000 93.5% chr5 + 134464794 134465143 350 browser details YourSeq 92 988 1110 3000 88.7% chr4 - 139627328 139627446 119 browser details YourSeq 92 989 1112 3000 88.4% chr1 - 151449671 151449788 118 browser details YourSeq 92 989 1112 3000 86.8% chr1 - 23542163 23542280 118 browser details YourSeq 91 989 1110 3000 89.4% chr18 + 38673209 38673326 118 browser details YourSeq 90 989 1111 3000 88.3% chr12 + 84116998 84117115 118 browser details YourSeq 90 989 1111 3000 89.2% chr1 + 128080207 128080324 118 browser details YourSeq 89 989 1113 3000 91.0% chr5 - 143951670 143951791 122 browser details YourSeq 89 986 1111 3000 89.2% chr9 + 96141664 96141785 122 browser details YourSeq 89 989 1112 3000 92.4% chr4 + 129283924 129284239 316 browser details YourSeq 89 993 1112 3000 89.2% chr1 + 39906546 39906661 116 browser details YourSeq 88 989 1112 3000 88.9% chr16 - 15822389 15822507 119 browser details YourSeq 88 989 1106 3000 87.5% chr1 - 120016733 120016846 114 browser details YourSeq 88 989 1102 3000 88.9% chr11 + 86750866 86750974 109

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% chr16 - 11196135 11199134 3000 browser details YourSeq 258 125 1436 3000 92.5% chr16 + 22205460 22328334 122875 browser details YourSeq 257 137 1436 3000 92.8% chr6 - 47959523 48012041 52519 browser details YourSeq 163 1200 1436 3000 91.0% chr15 + 93484418 93485035 618 browser details YourSeq 155 1247 1435 3000 91.4% chr16 + 5052285 5052475 191 browser details YourSeq 153 1246 1435 3000 90.6% chr13 - 94623204 94623394 191 browser details YourSeq 153 1247 1436 3000 91.0% chr16 + 20479868 20480070 203 browser details YourSeq 152 1245 1436 3000 90.9% chr13 - 97532363 97532560 198 browser details YourSeq 151 1247 1436 3000 90.4% chr9 + 53590271 53590466 196 browser details YourSeq 150 1264 1445 3000 91.8% chr18 - 35911926 35912117 192 browser details YourSeq 150 1264 1436 3000 93.7% chr4 + 57889250 57889430 181 browser details YourSeq 148 1267 1434 3000 94.1% chr1 + 185271918 185272085 168 browser details YourSeq 147 1260 1435 3000 92.1% chr1 - 160921865 160922044 180 browser details YourSeq 147 1255 1436 3000 91.6% chr18 + 62227373 62227557 185 browser details YourSeq 147 1268 1436 3000 93.5% chr12 + 33446346 33446514 169 browser details YourSeq 147 1264 1436 3000 93.1% chr10 + 77655099 77655275 177 browser details YourSeq 146 1262 1435 3000 92.5% chr12 - 59686012 59686186 175 browser details YourSeq 145 1267 1441 3000 91.5% chr8 - 13404403 13404577 175 browser details YourSeq 145 1260 1433 3000 92.0% chr2 + 101777581 101777757 177 browser details YourSeq 144 1256 1436 3000 87.8% chr9 + 65739718 65739890 173

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Rsl1d1 ribosomal L1 domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 66409, updated on 3-Jan-2021

Gene summary

Official Symbol Rsl1d1 provided by MGI Official Full Name ribosomal L1 domain containing 1 provided by MGI Primary source MGI:MGI:1913659 See related Ensembl:ENSMUSG00000005846 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as pBK1; C76439; 2410005K20Rik Expression Broad expression in CNS E11.5 (RPKM 40.8), liver E14 (RPKM 32.8) and 19 other tissues See more Orthologs human all NEW Try the new Data Table view

Genomic context

Location: 16; 16 A1 See Rsl1d1 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) 16 NC_000082.7 (11010901..11021156, complement)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (11193037..11203292, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (11193130..11203385, complement)

Chromosome 16 - NC_000082.7

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Transcript information: This gene has 5 transcripts

Gene: Rsl1d1 ENSMUSG00000005846

Description ribosomal L1 domain containing 1 [Source:MGI Symbol;Acc:MGI:1913659] Gene Synonyms 2410005K20Rik Location Chromosome 16: 11,192,970-11,203,331 reverse strand. GRCm38:CM001009.2 About this gene This gene has 5 transcripts (splice variants), 298 orthologues and 4 paralogues. Transcripts

UniProt Name Transcript ID bp Protein Translation ID Biotype CCDS Flags Match

Rsl1d1- ENSMUST00000119953.1 1945 452aa ENSMUSP00000113431.1 Protein coding CCDS49763 Q8BVY0 TSL:1 201 GENCODE basic APPRIS P1

Rsl1d1- ENSMUST00000230002.1 715 238aa ENSMUSP00000155495.1 Protein coding - A0A2R8VHW2 CDS 5' and 3' 204 incomplete

Rsl1d1- ENSMUST00000230232.1 415 33aa ENSMUSP00000155279.1 Nonsense mediated - A0A2R8VK65 CDS 5' 205 decay incomplete

Rsl1d1- ENSMUST00000129962.1 769 No - Retained intron - - TSL:2 203 protein

Rsl1d1- ENSMUST00000124454.1 764 No - Retained intron - - TSL:2 202 protein

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30.36 kb Forward strand 11.19Mb 11.20Mb 11.21Mb Genes 4930509G22Rik-201 >lincRNA 2610020C07Rik-201 >antisense (Comprehensive set...

2610020C07Rik-204 >antisense

2610020C07Rik-202 >antisense

2610020C07Rik-203 >antisense

Contigs < AC087541.23 Genes (Comprehensive set... < Gm7638-201processed pseudogene < Rsl1d1-201protein coding

< Gm6305-201processed pseudogen

< Rsl1d1-203retained intron < Rsl1d1-205nonsense mediated decay

< Rsl1d1-204protein coding

Regulatory Build

11.19Mb 11.20Mb 11.21Mb Reverse strand 30.36 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene pseudogene

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

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Transcript: ENSMUST00000119953

< Rsl1d1-201protein coding

Reverse strand 10.36 kb

ENSMUSP00000113... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Ribosomal protein L1-like Pfam Ribosomal protein L1/ribosomal biogenesis protein PANTHER PTHR23105

PTHR23105:SF31 Gene3D 3.30.190.20

Ribosomal protein L1, 3-layer alpha/beta-sandwich CDD Ribosomal protein L1/ribosomal biogenesis protein

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 452

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

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