https://www.alphaknockout.com

Mouse Aifm2 Knockout Project (CRISPR/Cas9)

Objective: To create a Aifm2 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Aifm2 (NCBI Reference Sequence: NM_001039194 ; Ensembl: ENSMUSG00000020085 ) is located on Mouse 10. 9 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000099706). Exon 4~8 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous null mice display reduced sensitivity to genotoxin induced cellular growth inhibition but have no change in spontaneous or induced tumor incidence.

Exon 4 starts from about 26.36% of the coding region. Exon 4~8 covers 60.41% of the coding region. The size of effective KO region: ~8012 bp. The KO region does not have any other known gene.

Page 1 of 9 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 7 8 9

Legends Exon of mouse Aifm2 Knockout region

Page 2 of 9 https://www.alphaknockout.com

Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1166 bp section upstream of Exon 4 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 924 bp section downstream of Exon 8 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

Page 3 of 9 https://www.alphaknockout.com

Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(1166bp) | A(21.78% 254) | C(24.87% 290) | T(28.9% 337) | G(24.44% 285)

Note: The 1166 bp section upstream of Exon 4 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(924bp) | A(31.06% 287) | C(23.7% 219) | T(23.81% 220) | G(21.43% 198)

Note: The 924 bp section downstream of Exon 8 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 4 of 9 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 1166 1 1166 1166 100.0% chr10 + 61726482 61727647 1166 browser details YourSeq 309 446 1138 1166 94.1% chr11 - 70310901 70556394 245494 browser details YourSeq 213 517 1137 1166 87.0% chr11 - 74709871 74710450 580 browser details YourSeq 211 513 1140 1166 84.6% chr4 + 123755405 123755838 434 browser details YourSeq 202 513 1144 1166 84.4% chr13 - 55288730 55289069 340 browser details YourSeq 188 546 1151 1166 95.7% chr4 + 133564756 133565371 616 browser details YourSeq 180 529 1137 1166 86.3% chr1 + 160922368 160922886 519 browser details YourSeq 171 933 1137 1166 94.3% chr1 + 88603387 88603591 205 browser details YourSeq 169 946 1140 1166 91.5% chr18 - 76812964 76813152 189 browser details YourSeq 168 940 1138 1166 95.2% chr4 - 40250567 40250765 199 browser details YourSeq 167 952 1155 1166 92.5% chr11 - 6609565 6609763 199 browser details YourSeq 167 615 1137 1166 85.7% chr11 + 23259836 23260036 201 browser details YourSeq 166 946 1154 1166 89.6% chr2 + 177532069 177532269 201 browser details YourSeq 166 946 1139 1166 94.6% chr11 + 88519804 88519996 193 browser details YourSeq 165 699 1138 1166 87.2% chr8 - 125661881 125662090 210 browser details YourSeq 163 952 1144 1166 95.6% chr13 - 74904202 74904395 194 browser details YourSeq 162 944 1139 1166 94.6% chr9 - 78067966 78068164 199 browser details YourSeq 162 952 1156 1166 91.3% chr9 - 72670784 72670984 201 browser details YourSeq 162 952 1140 1166 92.7% chr6 - 49447535 49447717 183 browser details YourSeq 161 622 1140 1166 85.5% chr2 - 32240168 32240358 191

Note: The 1166 bp section upstream of Exon 4 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 924 1 924 924 100.0% chr10 + 61735660 61736583 924 browser details YourSeq 136 136 313 924 92.1% chr10 + 117064448 117064957 510 browser details YourSeq 132 118 313 924 89.8% chr13 + 14046481 14046681 201 browser details YourSeq 130 134 305 924 89.7% chr13 + 104502258 104502445 188 browser details YourSeq 128 132 302 924 88.6% chr13 + 52493433 52493618 186 browser details YourSeq 127 134 298 924 93.3% chr10 + 61161469 61162044 576 browser details YourSeq 125 134 307 924 91.0% chr13 + 62431993 62432179 187 browser details YourSeq 123 133 298 924 89.2% chr1 + 186771755 186771936 182 browser details YourSeq 117 134 303 924 90.5% chr14 + 65141016 65141188 173 browser details YourSeq 116 156 311 924 93.4% chr13 + 64143690 64143859 170 browser details YourSeq 115 156 297 924 92.0% chr2 - 154068765 154068922 158 browser details YourSeq 115 134 297 924 90.8% chr10 + 122923478 122923652 175 browser details YourSeq 114 157 305 924 92.6% chr3 - 58502976 58503147 172 browser details YourSeq 113 156 298 924 91.8% chr11 - 86775011 86775163 153 browser details YourSeq 112 134 305 924 89.2% chr7 - 27959917 27960087 171 browser details YourSeq 111 149 295 924 89.6% chr11 - 84128298 84128461 164 browser details YourSeq 111 166 298 924 93.1% chr8 + 119772862 119773005 144 browser details YourSeq 107 149 304 924 92.3% chr13 - 54521605 54521798 194 browser details YourSeq 107 149 656 924 94.2% chr7 + 80800632 80801237 606 browser details YourSeq 107 133 289 924 92.2% chr4 + 133126741 133126904 164

Note: The 924 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 https://www.alphaknockout.com

Gene and information: Aifm2 -inducing factor, -associated 2 [ Mus musculus (house mouse) ] Gene ID: 71361, updated on 12-Aug-2019

Gene summary

Official Symbol Aifm2 provided by MGI Official Full Name apoptosis-inducing factor, mitochondrion-associated 2 provided by MGI Primary source MGI:MGI:1918611 See related Ensembl:ENSMUSG00000020085 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 Amid; PRG3; 5430437E11Rik; D730001I10Rik Expression Biased expression in adrenal adult (RPKM 65.8), bladder adult (RPKM 44.5) and 13 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 B4 See Aifm2 in Genome Data Viewer Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (61715263..61739260)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (61178011..61202008)

Chromosome 10 - NC_000076.6

Page 6 of 9 https://www.alphaknockout.com

Transcript information: This gene has 7 transcripts

Gene: Aifm2 ENSMUSG00000020085

Description apoptosis-inducing factor, mitochondrion-associated 2 [Source:MGI Symbol;Acc:MGI:1918611] Gene Synonyms 5430437E11Rik, Amid, D730001I10Rik, PRG3 Location : 61,715,263-61,739,260 forward strand. GRCm38:CM001003.2 About this gene This gene has 7 transcripts (splice variants), 204 orthologues, 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

Aifm2-203 ENSMUST00000099706.8 3086 373aa ENSMUSP00000097297.2 Protein coding CCDS23884 Q8BUE4 TSL:1 GENCODE basic APPRIS P1

Aifm2-204 ENSMUST00000105455.8 1512 373aa ENSMUSP00000101095.2 Protein coding CCDS23884 Q8BUE4 TSL:1 GENCODE basic APPRIS P1

Aifm2-201 ENSMUST00000067857.10 1442 373aa ENSMUSP00000070054.4 Protein coding CCDS23884 Q8BUE4 TSL:1 GENCODE basic APPRIS P1

Aifm2-202 ENSMUST00000080099.5 1329 380aa ENSMUSP00000078998.5 Protein coding CCDS48576 Q8BUE4 TSL:1 GENCODE basic

Aifm2-206 ENSMUST00000131361.1 656 No protein - Retained - - TSL:2

Aifm2-207 ENSMUST00000140664.1 477 No protein - Retained intron - - TSL:3

Aifm2-205 ENSMUST00000129809.1 378 No protein - lncRNA - - TSL:5

Page 7 of 9 https://www.alphaknockout.com

44.00 kb Forward strand 61.71Mb 61.72Mb 61.73Mb 61.74Mb (Comprehensive set... Aifm2-204 >protein coding

Aifm2-206 >retained intron Aifm2-207 >retained intron

Aifm2-201 >protein coding

Aifm2-203 >protein coding

Aifm2-205 >lncRNA Aifm2-202 >protein coding

Contigs AC153136.3 > Genes < H2afy2-201protein coding (Comprehensive set...

Regulatory Build

61.71Mb 61.72Mb 61.73Mb 61.74Mb Reverse strand 44.00 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

Page 8 of 9 https://www.alphaknockout.com

Transcript: ENSMUST00000099706

23.32 kb Forward strand

Aifm2-203 >protein coding

ENSMUSP00000097... Transmembrane heli... Low complexity (Seg) Superfamily FAD/NAD(P)-binding domain superfamily Prints PR00368

PR00469 Pfam FAD/NAD(P)-binding domain PANTHER PTHR43735:SF3

PTHR43735 Gene3D 3.50.50.100

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 373

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

Page 9 of 9