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Mouse Bcl2l11 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Bcl2l11 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Bcl2l11 gene (NCBI Reference Sequence: NM_207680 ; Ensembl: ENSMUSG00000027381 ) is located on Mouse chromosome 2. 4 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000110341). 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 Bcl2l11 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-134B6 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: Consistent with this gene's role in hematopoietic homeostasis, homozygous null mutants accumulate lymphoid and myeloid cells and succumb to autoimmune kidney disease.

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: 2367 bp, and the size of intron 2 for 3'-loxP site insertion: 18070 bp. The size of effective cKO region: ~882 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 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Bcl2l11 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(7382bp) | A(22.88% 1689) | C(24.71% 1824) | T(25.47% 1880) | G(26.94% 1989)

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% chr2 + 128125384 128128383 3000 browser details YourSeq 30 1205 1271 3000 96.9% chr12 - 21899334 21899573 240 browser details YourSeq 26 674 702 3000 96.5% chr13 - 46276310 46276339 30 browser details YourSeq 26 6 34 3000 96.6% chr10 - 50012252 50012281 30 browser details YourSeq 24 2320 2356 3000 83.4% chr2 + 30733584 30733621 38 browser details YourSeq 21 2952 2972 3000 100.0% chr1 - 188923279 188923299 21 browser details YourSeq 20 2947 2966 3000 100.0% chr1 - 58213949 58213968 20 browser details YourSeq 20 2935 2954 3000 100.0% chr1 + 9568464 9568483 20

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% chr2 + 128129266 128132265 3000 browser details YourSeq 31 2789 2821 3000 97.0% chr9 - 63816024 63816056 33 browser details YourSeq 24 2938 2963 3000 96.2% chr8 + 32323396 32323421 26 browser details YourSeq 23 869 894 3000 96.0% chr12 + 10369305 10369332 28

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

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Gene and protein information: Bcl2l11 BCL2-like 11 (apoptosis facilitator) [ Mus musculus (house mouse) ] Gene ID: 12125, updated on 10-Oct-2019

Gene summary

Official Symbol Bcl2l11 provided by MGI Official Full Name BCL2-like 11 (apoptosis facilitator) provided by MGI Primary source MGI:MGI:1197519 See related Ensembl:ENSMUSG00000027381 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 Bim; Bod; bcl2-L-11; 1500006F24Rik Expression Ubiquitous expression in thymus adult (RPKM 7.8), lung adult (RPKM 4.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 F1 See Bcl2l11 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (128126038..128162547)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (127951774..127988283)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 8 transcripts

Gene: Bcl2l11 ENSMUSG00000027381

Description BCL2-like 11 (apoptosis facilitator) [Source:MGI Symbol;Acc:MGI:1197519] Gene Synonyms 1500006F24Rik, Bim, Bod Location Chromosome 2: 128,126,038-128,162,547 forward strand. GRCm38:CM000995.2 About this gene This gene has 8 transcripts (splice variants), 103 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 74 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Bcl2l11- ENSMUST00000110341.8 5036 196aa ENSMUSP00000105970.2 Protein CCDS16713 O54918 Q542N5 TSL:1 205 coding GENCODE basic APPRIS P4

Bcl2l11- ENSMUST00000103210.1 3538 140aa ENSMUSP00000099499.1 Protein CCDS16714 O54918 TSL:1 203 coding GENCODE basic APPRIS ALT2

Bcl2l11- ENSMUST00000019281.13 2490 110aa ENSMUSP00000019281.7 Protein CCDS16712 O54918 TSL:1 201 coding Q3U7X3 GENCODE basic APPRIS ALT2

Bcl2l11- ENSMUST00000089634.11 2301 91aa ENSMUSP00000087062.5 Protein CCDS71141 Q6PEB3 TSL:1 202 coding GENCODE basic

Bcl2l11- ENSMUST00000103211.7 672 110aa ENSMUSP00000099500.1 Protein CCDS16712 O54918 TSL:2 204 coding Q3U7X3 GENCODE basic APPRIS ALT2

Bcl2l11- ENSMUST00000146023.7 1611 No - lncRNA - - TSL:2 208 protein

Bcl2l11- ENSMUST00000129206.1 974 No - lncRNA - - TSL:1 206 protein

Bcl2l11- ENSMUST00000132201.1 391 No - lncRNA - - TSL:2 207 protein

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56.51 kb Forward strand 128.12Mb 128.13Mb 128.14Mb 128.15Mb 128.16Mb 128.17Mb Genes (Comprehensive set... Acoxl-201 >protein coding Bcl2l11-201 >protein coding

Acoxl-202 >protein coding Bcl2l11-202 >protein coding

Bcl2l11-208 >lncRNA

Bcl2l11-205 >protein coding

Bcl2l11-204 >protein coding

Bcl2l11-203 >protein coding

Bcl2l11-206 >lncRNA

Bcl2l11-207 >lncRNA

Contigs AL805950.11 > Regulatory Build

128.12Mb 128.13Mb 128.14Mb 128.15Mb 128.16Mb 128.17Mb Reverse strand 56.51 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000110341

36.51 kb Forward strand

Bcl2l11-205 >protein coding

ENSMUSP00000105... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Pfam Apoptosis, Bim N-terminal Bcl-x interacting, BH3 domain

PIRSF Bcl-2-like protein 11

PANTHER PTHR12044:SF9

PTHR12044

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

Variant Legend synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 196

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

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