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Mouse Klrg1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Klrg1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Klrg1 gene (NCBI Reference Sequence: NM_016970 ; Ensembl: ENSMUSG00000030114 ) is located on Mouse chromosome 6. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000032207). 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 Klrg1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-260L19 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 allele have no abnormal phenoptype.

Exon 2 starts from about 14.72% 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: 2986 bp, and the size of intron 3 for 3'-loxP site insertion: 4481 bp. The size of effective cKO region: ~2129 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 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Klrg1 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(8629bp) | A(27.15% 2343) | C(20.3% 1752) | T(30.69% 2648) | G(21.86% 1886)

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% chr6 - 122279994 122282993 3000 browser details YourSeq 138 1457 1748 3000 89.8% chr6 - 51429778 51430164 387 browser details YourSeq 130 1448 1774 3000 85.4% chr14 + 61595086 61595647 562 browser details YourSeq 127 1455 1774 3000 88.3% chr11 + 102547639 102547953 315 browser details YourSeq 119 1455 1774 3000 87.4% chr8 - 109853824 109854181 358 browser details YourSeq 109 1411 1579 3000 91.7% chr5 - 40315090 40315395 306 browser details YourSeq 106 1450 1579 3000 90.8% chr4 + 89201514 89201643 130 browser details YourSeq 104 1466 1704 3000 87.2% chr2 - 119498944 119499424 481 browser details YourSeq 102 1455 1748 3000 91.9% chr7 - 30239049 30239412 364 browser details YourSeq 102 1307 1575 3000 88.8% chr2 - 96308085 96308355 271 browser details YourSeq 102 1447 1579 3000 88.7% chr1 - 30680296 30680429 134 browser details YourSeq 101 1455 1583 3000 90.0% chr4 - 119110476 119243716 133241 browser details YourSeq 101 1457 1579 3000 91.9% chr14 + 18762792 18762917 126 browser details YourSeq 100 1448 1579 3000 89.2% chr2 + 62525555 62525686 132 browser details YourSeq 99 1448 1579 3000 87.8% chr10 - 59961854 59961986 133 browser details YourSeq 98 1455 1579 3000 89.6% chr11 + 49688012 49688138 127 browser details YourSeq 97 1450 1582 3000 86.5% chr9 - 88602470 88602602 133 browser details YourSeq 97 1455 1579 3000 88.8% chr4 - 66989482 66989606 125 browser details YourSeq 97 1414 1572 3000 82.4% chr13 - 63803220 63803362 143 browser details YourSeq 97 1455 1579 3000 88.8% chr1 - 185347282 185347406 125

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 - 122274865 122277864 3000 browser details YourSeq 179 1644 2155 3000 95.5% chr5 + 118024745 118025267 523 browser details YourSeq 174 1631 1843 3000 92.4% chrX - 59354915 59355109 195 browser details YourSeq 170 1643 2146 3000 88.3% chr13 + 42858342 42858751 410 browser details YourSeq 169 1642 1843 3000 97.3% chr9 - 110705125 110807966 102842 browser details YourSeq 167 1627 1843 3000 92.2% chr17 - 70697117 70697319 203 browser details YourSeq 167 1644 1843 3000 97.2% chr10 - 108393604 108393810 207 browser details YourSeq 167 1643 1843 3000 92.7% chr7 + 24984136 24984320 185 browser details YourSeq 167 1644 1843 3000 93.8% chr2 + 29909494 29909683 190 browser details YourSeq 166 1642 1843 3000 92.2% chrX - 101486852 101487037 186 browser details YourSeq 166 1641 1838 3000 93.1% chr2 - 131451788 131451967 180 browser details YourSeq 166 1642 1843 3000 92.3% chr1 - 151453667 151453855 189 browser details YourSeq 166 1645 1843 3000 96.1% chr1 - 133022385 133022582 198 browser details YourSeq 166 1645 1843 3000 97.8% chr5 + 90410339 90410553 215 browser details YourSeq 166 1644 1843 3000 93.7% chr19 + 18748991 18749174 184 browser details YourSeq 165 1644 1843 3000 92.6% chr19 - 3491858 3492042 185 browser details YourSeq 165 1643 1843 3000 92.1% chr12 - 76638320 76638504 185 browser details YourSeq 165 1649 1843 3000 93.2% chr1 - 74557325 74557504 180 browser details YourSeq 165 1642 1843 3000 92.3% chr13 + 93543230 93543423 194 browser details YourSeq 165 1648 1843 3000 92.0% chr1 + 60459167 60459343 177

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 protein information: Klrg1 killer cell lectin-like receptor subfamily G, member 1 [ Mus musculus (house mouse) ] Gene ID: 50928, updated on 1-Oct-2019

Gene summary

Official Symbol Klrg1 provided by MGI Official Full Name killer cell lectin-like receptor subfamily G, member 1 provided by MGI Primary source MGI:MGI:1355294 See related Ensembl:ENSMUSG00000030114 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 MAFA; 2F1-Ag; MAFA-L Expression Low expression observed in reference dataset See more Orthologs human all

Genomic context

Location: 6 F1; 6 57.52 cM See Klrg1 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (122270596..122282833, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (122220614..122232851, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 1 transcript

Gene: Klrg1 ENSMUSG00000030114

Description killer cell lectin-like receptor subfamily G, member 1 [Source:MGI Symbol;Acc:MGI:1355294] Gene Synonyms 2F1-Ag Location Chromosome 6: 122,270,596-122,282,882 reverse strand. GRCm38:CM000999.2 About this gene This gene has 1 transcript (splice variant), 109 orthologues, 44 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Klrg1-201 ENSMUST00000032207.8 1414 188aa ENSMUSP00000032207.8 Protein coding CCDS20492 O88713 TSL:1 GENCODE basic APPRIS P1

32.29 kb Forward strand

122.27Mb 122.28Mb 122.29Mb Contigs < AC153582.10 AC153579.10 > Genes (Comprehensive set... < Klrg1-201protein coding

Regulatory Build

122.27Mb 122.28Mb 122.29Mb Reverse strand 32.29 kb

Regulation Legend

CTCF Enhancer Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

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Transcript: ENSMUST00000032207

< Klrg1-201protein coding

Reverse strand 12.29 kb

ENSMUSP00000032... Transmembrane heli... PDB-ENSP mappings Superfamily C-type lectin fold

SMART C-type lectin-like

Pfam C-type lectin-like

PROSITE profiles C-type lectin-like PANTHER Killer cell lectin-like receptor subfamily G member 1

Gene3D C-type lectin-like/link domain superfamily CDD Natural killer cell receptor-like, C-type lectin-like domain

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

Variant Legend

missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 188

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

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