https://www.alphaknockout.com

Mouse Lrrc8e Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Lrrc8e conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Lrrc8e gene (NCBI Reference Sequence: NM_028175 ; Ensembl: ENSMUSG00000046589 ) is located on Mouse chromosome 8. 3 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000053035). 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 Lrrc8e gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-463K24 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 100% 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: 4656 bp, and the size of intron 2 for 3'-loxP site insertion: 2110 bp. The size of effective cKO region: ~638 bp. The cKO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

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 Lrrc8e Homology arm cKO region loxP site

Page 2 of 8 https://www.alphaknockout.com

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(7138bp) | A(25.55% 1824) | C(24.84% 1773) | T(25.71% 1835) | G(23.9% 1706)

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.

Page 3 of 8 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 + 4228418 4231417 3000 browser details YourSeq 379 318 1277 3000 88.4% chr5 + 149444728 149652269 207542 browser details YourSeq 258 314 1282 3000 88.6% chr6 - 49308574 49647370 338797 browser details YourSeq 249 290 788 3000 83.9% chr4 + 124014169 124014660 492 browser details YourSeq 220 312 802 3000 83.4% chr11 + 3891000 3891479 480 browser details YourSeq 201 292 793 3000 85.4% chr6 - 94778374 94778886 513 browser details YourSeq 197 292 793 3000 83.1% chr2 - 34209359 34209839 481 browser details YourSeq 197 1086 1313 3000 93.8% chr10 - 25337264 25337554 291 browser details YourSeq 187 316 776 3000 82.9% chr8 + 126403690 126404153 464 browser details YourSeq 186 1111 1314 3000 94.9% chr5 - 114836225 114836422 198 browser details YourSeq 186 292 776 3000 82.4% chr8 + 64620499 64620947 449 browser details YourSeq 185 382 764 3000 81.8% chrX + 92578709 92579120 412 browser details YourSeq 184 1106 1306 3000 97.5% chr9 + 21118988 21119314 327 browser details YourSeq 184 1074 1293 3000 95.6% chr4 + 116414633 116415034 402 browser details YourSeq 183 1082 1315 3000 93.4% chr17 - 74524725 74525053 329 browser details YourSeq 183 1124 1326 3000 93.9% chr17 + 21846506 21846702 197 browser details YourSeq 181 1123 1311 3000 98.5% chr10 - 128052108 128052652 545 browser details YourSeq 179 1107 1311 3000 92.8% chr8 - 107111599 107111792 194 browser details YourSeq 179 1123 1312 3000 95.8% chr15 - 79939751 79939938 188 browser details YourSeq 178 1122 1315 3000 96.9% chrX - 100513019 100513214 196

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% chr8 + 4232056 4235055 3000 browser details YourSeq 182 1209 1669 3000 84.5% chr7 + 27312375 27312738 364 browser details YourSeq 162 67 230 3000 99.4% chr8 + 84016593 84016756 164 browser details YourSeq 162 67 230 3000 99.4% chr8 + 69766984 69767147 164 browser details YourSeq 161 53 252 3000 94.5% chr9 + 65778183 65778392 210 browser details YourSeq 160 67 230 3000 98.8% chr9 - 64280705 64280868 164 browser details YourSeq 160 66 230 3000 98.8% chr1 - 183377845 183378013 169 browser details YourSeq 159 68 230 3000 98.8% chr1 + 23901974 23902136 163 browser details YourSeq 157 68 230 3000 98.2% chr3 - 79139804 79139966 163 browser details YourSeq 157 68 230 3000 98.2% chr10 - 35284153 35284315 163 browser details YourSeq 157 68 251 3000 95.4% chr7 + 141021801 141022126 326 browser details YourSeq 157 68 230 3000 98.2% chr10 + 67199511 67199673 163 browser details YourSeq 156 68 227 3000 98.8% chr2 + 26136116 26136275 160 browser details YourSeq 156 68 223 3000 100.0% chr1 + 9751416 9751571 156 browser details YourSeq 155 67 229 3000 97.6% chr14 - 51013960 51014122 163 browser details YourSeq 154 52 241 3000 89.6% chr1 + 84859970 84860147 178 browser details YourSeq 153 68 230 3000 97.0% chr9 - 10937579 10937741 163 browser details YourSeq 151 68 222 3000 98.8% chr7 - 107734127 107734281 155 browser details YourSeq 151 68 230 3000 96.4% chr7 - 15965433 15965595 163 browser details YourSeq 150 67 226 3000 96.9% chrX + 106172726 106172885 160

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

Page 4 of 8 https://www.alphaknockout.com

Gene and protein information: Lrrc8e leucine rich repeat containing 8 family, member E [ Mus musculus (house mouse) ] Gene ID: 72267, updated on 10-Oct-2019

Gene summary

Official Symbol Lrrc8e provided by MGI Official Full Name leucine rich repeat containing 8 family, member E provided by MGI Primary source MGI:MGI:1919517 See related Ensembl:ENSMUSG00000046589 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 C87354; 1810049O03Rik Expression Biased expression in adrenal adult (RPKM 4.6), bladder adult (RPKM 3.6) and 8 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 A1.1 See Lrrc8e in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (4224268..4237470)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (4226827..4237470)

Chromosome 8 - NC_000074.6

Page 5 of 8 https://www.alphaknockout.com

Transcript information: This gene has 2 transcripts

Gene: Lrrc8e ENSMUSG00000046589

Description leucine rich repeat containing 8 family, member E [Source:MGI Symbol;Acc:MGI:1919517] Gene Synonyms 1810049O03Rik, C87354 Location Chromosome 8: 4,226,827-4,237,470 forward strand. GRCm38:CM001001.2 About this gene This gene has 2 transcripts (splice variants), 203 orthologues, 33 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Lrrc8e-201 ENSMUST00000053035.6 3878 795aa ENSMUSP00000052055.6 Protein coding CCDS22079 Q66JT1 TSL:1 GENCODE basic APPRIS P1

Lrrc8e-202 ENSMUST00000207770.1 455 64aa ENSMUSP00000146637.1 Protein coding - A0A140LI19 CDS 3' incomplete TSL:3

Page 6 of 8 https://www.alphaknockout.com

30.64 kb Forward strand 4.22Mb 4.23Mb 4.24Mb Genes (Comprehensive set... Lrrc8e-201 >protein coding Map2k7-206 >protein coding

Lrrc8e-202 >protein coding Map2k7-202 >protein coding

Map2k7-207 >protein coding

Map2k7-201 >protein coding

Gm49320-201 >nonsense mediated decay

Map2k7-204 >protein coding

Map2k7-203 >protein coding

Map2k7-208 >retained intron

Map2k7-205 >protein coding

Map2k7-209 >protein coding

Map2k7-210 >lncRNA

Contigs AC123029.3 > Genes < Prr36-201protein coding (Comprehensive set...

< Prr36-204protein coding

< Prr36-202protein coding

< Prr36-203protein coding

Regulatory Build

4.22Mb 4.23Mb 4.24Mb Reverse strand 30.64 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000053035

10.64 kb Forward strand

Lrrc8e-201 >protein coding

ENSMUSP00000052... Transmembrane heli... Low complexity (Seg) Superfamily SSF52058 SMART Leucine-rich repeat, typical subtype

SM00364 Pfam LRRC8, pannexin-like TM region Leucine-rich repeat

PROSITE profiles Leucine-rich repeat PANTHER PTHR45752:SF8

PTHR45752 Gene3D Leucine-rich repeat domain superfamily

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

Variant Legend missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 795

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

Page 8 of 8