https://www.alphaknockout.com

Mouse Hars2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Hars2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Hars2 gene (NCBI Reference Sequence: NM_080636 ; Ensembl: ENSMUSG00000019143 ) is located on Mouse chromosome 18. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000152954). Exon 5~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Hars2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-56M5 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 5 starts from about 26.2% of the coding region. The knockout of Exon 5~8 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1295 bp, and the size of intron 8 for 3'-loxP site insertion: 486 bp. The size of effective cKO region: ~1662 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 5' gRNA region gRNA region 3'

1 2 3 4 5 6 7 8 9 10 11 12 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Hars2 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8155bp) | A(25.05% 2043) | C(19.67% 1604) | T(30.96% 2525) | G(24.32% 1983)

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% chr18 + 36784253 36787252 3000 browser details YourSeq 223 498 1078 3000 88.5% chr11 - 72555350 72555711 362 browser details YourSeq 221 558 1081 3000 90.1% chr16 + 17231542 17231988 447 browser details YourSeq 208 558 1063 3000 88.6% chr8 - 13970336 13970601 266 browser details YourSeq 198 646 1081 3000 87.9% chr7 + 27630398 27630785 388 browser details YourSeq 197 558 1063 3000 85.1% chr14 - 63518559 63518828 270 browser details YourSeq 195 499 1064 3000 91.5% chr7 + 28397022 28397597 576 browser details YourSeq 191 873 1080 3000 94.5% chr9 + 6237817 6238014 198 browser details YourSeq 187 873 1063 3000 99.0% chr6 + 47857719 47857909 191 browser details YourSeq 186 877 1078 3000 97.0% chr7 + 31493942 31494142 201 browser details YourSeq 184 558 1063 3000 86.1% chr5 - 121563921 121564167 247 browser details YourSeq 183 873 1063 3000 98.0% chr8 - 119180199 119180389 191 browser details YourSeq 183 873 1063 3000 98.0% chr3 - 10432318 10432508 191 browser details YourSeq 183 873 1063 3000 96.9% chr9 + 114502463 114502652 190 browser details YourSeq 183 873 1063 3000 96.9% chr6 + 113175930 113176119 190 browser details YourSeq 183 872 1063 3000 98.0% chr11 + 49810673 49810866 194 browser details YourSeq 182 876 1063 3000 98.5% chr4 + 132798935 132799122 188 browser details YourSeq 182 875 1063 3000 96.8% chr3 + 88154076 88154262 187 browser details YourSeq 182 873 1063 3000 98.0% chr18 + 56582308 56582502 195 browser details YourSeq 180 871 1080 3000 95.1% chr2 - 167064436 167064649 214

Note: The 3000 bp section upstream of Exon 5 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% chr18 + 36788915 36791914 3000 browser details YourSeq 1151 248 3000 3000 88.1% chrX + 59196963 59198442 1480 browser details YourSeq 111 828 2995 3000 84.3% chr1 + 85547364 85914416 367053 browser details YourSeq 77 2 176 3000 88.9% chr10 - 101476053 101476237 185 browser details YourSeq 65 834 1037 3000 84.1% chr16 - 44147085 44147271 187 browser details YourSeq 64 66 159 3000 82.5% chr5 - 125234758 125234837 80 browser details YourSeq 64 837 1034 3000 91.1% chr17 - 4600656 4600941 286 browser details YourSeq 64 836 1034 3000 87.5% chr15 + 99286100 99286297 198 browser details YourSeq 63 42 121 3000 90.0% chr6 - 31239203 31239287 85 browser details YourSeq 62 73 170 3000 90.6% chr2 - 74641666 74641817 152 browser details YourSeq 61 80 176 3000 78.5% chr1 - 141109337 141109425 89 browser details YourSeq 60 839 1037 3000 79.5% chr11 - 6465115 6465283 169 browser details YourSeq 59 827 1035 3000 77.5% chr1 - 59694496 59694687 192 browser details YourSeq 58 837 1034 3000 94.0% chr11 - 48777724 48778067 344 browser details YourSeq 55 1 151 3000 92.5% chr10 - 4343530 4344078 549 browser details YourSeq 53 1006 1166 3000 92.1% chr1 + 153505882 153506156 275 browser details YourSeq 50 828 1036 3000 87.0% chr10 - 61243822 61244149 328 browser details YourSeq 49 968 1036 3000 98.1% chr10 - 56346906 56347277 372 browser details YourSeq 48 2916 2991 3000 81.6% chr4 - 149816304 149816379 76 browser details YourSeq 48 2913 2990 3000 80.8% chr1 - 16558404 16558481 78

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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

Gene and protein information: Hars2 histidyl-tRNA synthetase 2 [ Mus musculus (house mouse) ] Gene ID: 70791, updated on 12-Aug-2019

Gene summary

Official Symbol Hars2 provided by MGI Official Full Name histidyl-tRNA synthetase 2 provided by MGI Primary source MGI:MGI:1918041 See related Ensembl:ENSMUSG00000019143 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as HO3; HARSR; Harsl; 4631412B19Rik Summary This gene encodes a putative member of the class II family of aminoacyl-tRNA synthetases. These enzymes play a critical Expression role in protein biosynthesis by charging tRNAs with their cognate amino acids. This protein is encoded by the nuclear genome but is likely to be imported to the mitochondrion where it is thought to catalyze the ligation of histidine to tRNA molecules. Mutations in a similar gene in human have been associated with Perrault syndrome 2 (PRLTS2). [provided by RefSeq, Mar 2015] Orthologs Ubiquitous expression in CNS E11.5 (RPKM 11.8), CNS E14 (RPKM 10.4) and 28 other tissues See more human all

Genomic context

Location: 18; 18 B2 See Hars2 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (36783202..36792562)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (36942934..36952216)

Chromosome 18 - NC_000084.6

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

Transcript information: This gene has 7 transcripts

Gene: Hars2 ENSMUSG00000019143

Description histidyl-tRNA synthetase 2 [Source:MGI Symbol;Acc:MGI:1918041] Gene Synonyms 4631412B19Rik, HARSR, HO3, Harsl Location Chromosome 18: 36,783,008-36,792,562 forward strand. GRCm38:CM001011.2 About this gene This gene has 7 transcripts (splice variants), 225 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Hars2-206 ENSMUST00000152954.7 3336 505aa ENSMUSP00000117231.1 Protein coding CCDS29165 Q99KK9 TSL:1 GENCODE basic APPRIS P1

Hars2-201 ENSMUST00000019287.8 2002 424aa ENSMUSP00000019287.8 Protein coding CCDS84376 G5E823 TSL:1 GENCODE basic

Hars2-203 ENSMUST00000131952.1 819 No protein - Retained intron - - TSL:5

Hars2-205 ENSMUST00000145876.1 772 No protein - Retained intron - - TSL:2

Hars2-202 ENSMUST00000124204.1 769 No protein - Retained intron - - TSL:1

Hars2-207 ENSMUST00000155842.1 406 No protein - Retained intron - - TSL:5

Hars2-204 ENSMUST00000134122.7 896 No protein - lncRNA - - TSL:3

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

29.55 kb Forward strand 36.78Mb 36.79Mb 36.80Mb Genes Hars2-206 >protein coding Zmat2-201 >protein coding (Comprehensive set...

Hars2-204 >lncRNA Zmat2-202 >retained intron

Hars2-201 >protein coding Vaultrc5-201 >misc RNA

Hars2-205 >retained intron Hars2-207 >retained intron

Hars2-203 >retained intron

Hars2-202 >retained intron

Contigs AC027740.11 > Genes < Hars-201protein coding (Comprehensive set...

< Hars-204retained intron

< Hars-203nonsense mediated decay

Regulatory Build

36.78Mb 36.79Mb 36.80Mb Reverse strand 29.55 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

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

Transcript: ENSMUST00000152954

9.55 kb Forward strand

Hars2-206 >protein coding

ENSMUSP00000117... Low complexity (Seg) Cleavage site (Sign... TIGRFAM Histidine-tRNA ligase

Superfamily SSF55681 SSF52954

Pfam Class II Histidinyl-tRNA synthetase (HisRS)-like catalytic core domain Anticodon-binding

PROSITE profiles Aminoacyl-tRNA synthetase, class II

PIRSF Histidine-tRNA ligase/ATP phosphoribosyltransferase regulatory subunit

PANTHER PTHR11476

PTHR11476:SF6 Gene3D 3.30.930.10 Anticodon-binding domain superfamily

CDD Class II Histidinyl-tRNA synthetase (HisRS)-like catalytic core domain Histidyl-anticodon-binding

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 505

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

Page 8 of 8