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Mouse Actl6b Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Actl6b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Actl6b gene (NCBI Reference Sequence: NM_031404 ; Ensembl: ENSMUSG00000029712 ) is located on Mouse chromosome 5. 14 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000139395). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Actl6b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-367M19 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: Homozygotes for null mutations exhibit low survivor rate and most die within 2 days after birth and show hyperactivity due to reduced dendrite formation in neurons.

Exon 5 starts from about 28.95% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 8010 bp, and the size of intron 5 for 3'-loxP site insertion: 1286 bp. The size of effective cKO region: ~598 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 5 6 7 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Actl6b 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. 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(7098bp) | A(26.32% 1868) | C(22.57% 1602) | T(26.29% 1866) | G(24.82% 1762)

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% chr5 + 137559858 137562857 3000 browser details YourSeq 349 1 1742 3000 90.3% chr11 - 94706811 94900336 193526 browser details YourSeq 175 1460 1744 3000 88.7% chr8 - 106775721 106776021 301 browser details YourSeq 161 1435 1704 3000 90.9% chr19 - 4002342 4002607 266 browser details YourSeq 148 1479 1753 3000 92.1% chr15 + 76279860 76280341 482 browser details YourSeq 143 1435 1592 3000 96.2% chr10 + 6767930 6768106 177 browser details YourSeq 140 1435 1592 3000 95.0% chr11 + 55138820 55139047 228 browser details YourSeq 139 1065 1592 3000 83.2% chr10 - 4226035 4226193 159 browser details YourSeq 137 1435 1592 3000 95.5% chr15 + 26871585 26871742 158 browser details YourSeq 134 1435 1584 3000 95.4% chr15 + 93363811 93363961 151 browser details YourSeq 134 1435 1592 3000 92.3% chr11 + 60081208 60081364 157 browser details YourSeq 133 1429 1592 3000 90.6% chr12 - 118291258 118291420 163 browser details YourSeq 133 1067 1592 3000 81.2% chr11 - 26856006 26856176 171 browser details YourSeq 127 1435 1592 3000 92.3% chr11 - 33183095 33183258 164 browser details YourSeq 126 1436 1585 3000 94.4% chr11 - 50165781 50166094 314 browser details YourSeq 126 1435 1592 3000 88.2% chr13 + 114663162 114663306 145 browser details YourSeq 126 1444 1592 3000 91.0% chr12 + 34344200 34344345 146 browser details YourSeq 126 1435 1592 3000 88.4% chr1 + 13400833 13400982 150 browser details YourSeq 125 1436 1592 3000 89.2% chr1 - 37461945 37462097 153 browser details YourSeq 125 1435 1592 3000 88.1% chr1 + 180655331 180655472 142

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% chr5 + 137563456 137566455 3000 browser details YourSeq 98 204 570 3000 83.2% chr17 + 74748367 74748671 305 browser details YourSeq 97 1068 1298 3000 84.4% chr3 + 32718063 32718468 406 browser details YourSeq 90 1506 2465 3000 94.2% chr3 - 13245343 13371958 126616 browser details YourSeq 84 1068 1298 3000 72.7% chr2 + 68082151 68082314 164 browser details YourSeq 80 1507 1736 3000 89.9% chr5 - 129492452 129492762 311 browser details YourSeq 72 207 557 3000 89.7% chr8 + 33327786 33328135 350 browser details YourSeq 63 1503 1621 3000 87.1% chr7 - 83855736 83875596 19861 browser details YourSeq 62 1509 1873 3000 72.8% chr11 - 69114933 69115087 155 browser details YourSeq 62 1506 1621 3000 93.1% chr18 + 84591193 84591697 505 browser details YourSeq 60 218 822 3000 72.4% chr7 + 34803863 34804228 366 browser details YourSeq 59 1506 1663 3000 92.8% chr13 + 11656176 11656466 291 browser details YourSeq 58 1506 1621 3000 90.3% chrX - 141850267 141850386 120 browser details YourSeq 54 537 1560 3000 84.7% chr12 - 80831930 80995821 163892 browser details YourSeq 52 1506 1866 3000 66.2% chr2 - 153296846 153297001 156 browser details YourSeq 50 1504 1596 3000 79.0% chr7 + 137035203 137035293 91 browser details YourSeq 50 1504 1588 3000 90.4% chr7 + 3427765 3428159 395 browser details YourSeq 50 803 862 3000 91.7% chr5 + 8147323 8147382 60 browser details YourSeq 49 223 823 3000 60.4% chr1 - 172166710 172166776 67 browser details YourSeq 49 499 830 3000 67.3% chr2 + 147099221 147099401 181

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Actl6b actin-like 6B [ Mus musculus (house mouse) ] Gene ID: 83766, updated on 15-Oct-2019

Gene summary

Official Symbol Actl6b provided by MGI Official Full Name actin-like 6B provided by MGI Primary source MGI:MGI:1933548 See related Ensembl:ENSMUSG00000029712 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 Actl6; ArpNa; Baf53b Expression Biased expression in CNS E18 (RPKM 68.3), whole brain E14.5 (RPKM 63.6) and 5 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 G2 See Actl6b in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (137553361..137569582)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (137994783..138010801)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 9 transcripts

Gene: Actl6b ENSMUSG00000029712

Description actin-like 6B [Source:MGI Symbol;Acc:MGI:1933548] Gene Synonyms Actl6, ArpNa, Baf53b Location Chromosome 5: 137,553,517-137,569,582 forward strand. GRCm38:CM000998.2 About this gene This gene has 9 transcripts (splice variants), 119 orthologues, 27 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Actl6b- ENSMUST00000139395.7 1592 426aa ENSMUSP00000119356.1 Protein coding CCDS19770 Q99MR0 TSL:1 205 GENCODE basic APPRIS P1

Actl6b- ENSMUST00000136565.7 633 116aa ENSMUSP00000117425.1 Protein coding - D3Z4L2 CDS 3' 204 incomplete TSL:3

Actl6b- ENSMUST00000136088.7 508 74aa ENSMUSP00000117138.1 Protein coding - D3Z5W4 CDS 3' 203 incomplete TSL:3

Actl6b- ENSMUST00000149292.7 471 8aa ENSMUSP00000123004.1 Protein coding - A0A1C7ZN05 CDS 3' 207 incomplete TSL:3

Actl6b- ENSMUST00000125489.1 432 20aa ENSMUSP00000121672.1 Protein coding - D3Z2E2 CDS 3' 202 incomplete TSL:3

Actl6b- ENSMUST00000031725.9 1549 381aa ENSMUSP00000031725.8 Nonsense mediated - F8WI57 TSL:5 201 decay

Actl6b- ENSMUST00000198601.1 689 65aa ENSMUSP00000143702.1 Nonsense mediated - A0A0G2JGU5 TSL:2 209 decay

Actl6b- ENSMUST00000196154.1 3100 No - Retained intron - - TSL:NA 208 protein

Actl6b- ENSMUST00000145500.1 405 No - Retained intron - - TSL:2 206 protein

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36.07 kb Forward strand 137.55Mb 137.56Mb 137.57Mb Genes (Comprehensive set... Actl6b-203 >protein coding Tfr2-207 >protein coding

Actl6b-205 >protein coding Tfr2-201 >protein coding

Actl6b-209 >nonsense mediated decay Tfr2-211 >retained intron

Actl6b-206 >retained intron Tfr2-208 >protein coding

Actl6b-204 >protein coding Tfr2-204 >protein coding

Actl6b-202 >protein coding Tfr2-206 >protein coding

Actl6b-207 >protein coding Tfr2-210 >retained intron

Actl6b-201 >nonsense mediated decay Tfr2-205 >retained intron

Actl6b-208 >retained intron Tfr2-202 >protein coding

Contigs AC148018.3 > Regulatory Build

137.55Mb 137.56Mb 137.57Mb Reverse strand 36.07 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000139395

16.06 kb Forward strand

Actl6b-205 >protein coding

ENSMUSP00000119... Low complexity (Seg) Superfamily SSF53067 SMART Actin family

Prints Actin family Pfam Actin family

PROSITE patterns Actin, conserved site

PANTHER Actin family

PTHR11937:SF196 Gene3D 3.30.420.40

3.90.640.10 CDD cd00012

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

Variant Legend

synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 426

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

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