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Mouse Mrpl19 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mrpl19 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mrpl19 (NCBI Reference Sequence: NM_026490 ; Ensembl: ENSMUSG00000030045 ) is located on Mouse 6. 5 exons are identified, with the ATG in exon 1 and the TGA in exon 5 (Transcript: ENSMUST00000032124). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mrpl19 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-271I13 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 25.34% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1349 bp, and the size of intron 3 for 3'-loxP site insertion: 1436 bp. The size of effective cKO region: ~924 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 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Mrpl19 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(7424bp) | A(26.51% 1968) | C(21.94% 1629) | T(27.3% 2027) | G(24.25% 1800)

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% chr6 - 81964608 81967607 3000 browser details YourSeq 228 200 555 3000 84.8% chr4 + 137934726 137935045 320 browser details YourSeq 190 194 528 3000 81.7% chr8 - 126241902 126242220 319 browser details YourSeq 190 280 526 3000 89.4% chr5 - 149267930 149393543 125614 browser details YourSeq 186 209 501 3000 83.0% chr5 + 112139403 112139714 312 browser details YourSeq 178 211 556 3000 78.1% chr14 + 27954768 27955111 344 browser details YourSeq 177 291 558 3000 83.7% chr17 - 46322962 46323228 267 browser details YourSeq 176 199 478 3000 81.6% chr1 - 125498263 125498542 280 browser details YourSeq 175 193 501 3000 79.7% chr1 - 139163585 139163889 305 browser details YourSeq 175 280 558 3000 86.5% chr15 + 38703100 38703375 276 browser details YourSeq 173 150 547 3000 81.7% chr8 + 24929262 24929742 481 browser details YourSeq 173 209 558 3000 81.1% chr1 + 171661943 171662275 333 browser details YourSeq 171 204 466 3000 82.8% chr5 + 111517571 111517832 262 browser details YourSeq 170 280 554 3000 82.0% chr7 + 112064762 112065035 274 browser details YourSeq 169 223 558 3000 77.9% chr16 - 8316770 8317094 325 browser details YourSeq 168 210 470 3000 82.4% chr11 + 5806979 5807241 263 browser details YourSeq 167 200 484 3000 79.9% chr17 - 68215101 68215383 283 browser details YourSeq 167 212 467 3000 82.9% chr12 - 106550327 106550583 257 browser details YourSeq 166 274 558 3000 83.8% chr2 + 18630861 18631126 266 browser details YourSeq 166 309 552 3000 84.6% chr1 + 13610947 13611187 241

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% chr6 - 81960684 81963683 3000 browser details YourSeq 166 2564 2844 3000 88.8% chr12 + 54469416 54469762 347 browser details YourSeq 161 2565 2881 3000 85.5% chr1 - 4956761 4957068 308 browser details YourSeq 160 1932 2097 3000 98.2% chr17 + 29454086 29454251 166 browser details YourSeq 158 2560 2863 3000 86.3% chr8 + 115447415 115447771 357 browser details YourSeq 153 2571 2860 3000 86.3% chr5 - 129453475 129453795 321 browser details YourSeq 153 1929 2095 3000 95.9% chr2 - 31737025 31737191 167 browser details YourSeq 151 1929 2089 3000 96.9% chr1 - 71718876 71719036 161 browser details YourSeq 149 1928 2099 3000 93.7% chr12 + 12955320 12955492 173 browser details YourSeq 147 2559 2862 3000 85.5% chr7 - 24123067 24123365 299 browser details YourSeq 147 1929 2098 3000 90.9% chr1 - 53077038 53077201 164 browser details YourSeq 144 2565 2862 3000 86.8% chr9 - 102703217 102703531 315 browser details YourSeq 144 1936 2087 3000 97.4% chr4 + 126988318 126988469 152 browser details YourSeq 143 1929 2091 3000 93.9% chr1 + 133112516 133112678 163 browser details YourSeq 142 2557 2856 3000 89.1% chr10 - 7978633 7978952 320 browser details YourSeq 141 1929 2092 3000 90.4% chr3 - 41137036 41137191 156 browser details YourSeq 139 2566 2859 3000 84.8% chr16 + 84722227 84722515 289 browser details YourSeq 138 2572 2860 3000 86.4% chr1 - 20992892 20993201 310 browser details YourSeq 136 2593 2861 3000 89.1% chr13 - 53645998 53646277 280 browser details YourSeq 136 2606 2862 3000 87.3% chr19 + 29155130 29155401 272

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: Mrpl19 mitochondrial L19 [ Mus musculus (house mouse) ] Gene ID: 56284, updated on 12-Aug-2019

Gene summary

Official Symbol Mrpl19 provided by MGI Official Full Name mitochondrial ribosomal protein L19 provided by MGI Primary source MGI:MGI:1926274 See related Ensembl:ENSMUSG00000030045 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 RLX1; Rpml15; MRP-L15; D6Ertd157e; 9030416F12Rik; 9130412E02Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 6.3), CNS E18 (RPKM 4.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: 6 C3; 6 35.81 cM See Mrpl19 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (81957826..81965949, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (81907820..81915943, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 3 transcripts

Gene: Mrpl19 ENSMUSG00000030045

Description mitochondrial ribosomal protein L19 [Source:MGI Symbol;Acc:MGI:1926274] Gene Synonyms 9030416F12Rik, D6Ertd157e, MRP-L15, RLX1, Rpml15 Location Chromosome 6: 81,957,851-81,965,958 reverse strand. GRCm38:CM000999.2 About this gene This gene has 3 transcripts (splice variants), 202 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein ID Biotype CCDS UniProt Flags

Mrpl19-201 ENSMUST00000032124.8 5000 292aa ENSMUSP00000032124.8 Protein coding CCDS39523 Q9D338 TSL:1 GENCODE basic APPRIS P1

Mrpl19-202 ENSMUST00000128374.1 732 No protein - Retained intron - - TSL:2

Mrpl19-203 ENSMUST00000148025.1 419 No protein - lncRNA - - TSL:3

28.11 kb Forward strand 81.95Mb 81.96Mb 81.97Mb Gcfc2-201 >protein coding (Comprehensive set...

Gcfc2-206 >nonsense mediated decay

Gcfc2-207 >protein coding

Gcfc2-203 >retained intron

Contigs < AC129024.4 Genes (Comprehensive set... < Mrpl19-201protein coding

< Mrpl19-202retained intron

< Mrpl19-203lncRNA

Regulatory Build

81.95Mb 81.96Mb 81.97Mb Reverse strand 28.11 kb

Regulation Legend

CTCF 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: ENSMUST00000032124

< Mrpl19-201protein coding

Reverse strand 8.11 kb

ENSMUSP00000032... Low complexity (Seg) Superfamily Translation protein SH3-like domain superfamily Prints Ribosomal protein L19 Pfam Ribosomal protein L19 PANTHER PTHR15680:SF9

Ribosomal protein L19 Gene3D Ribosomal protein L19 superfamily

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 292

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

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