Exceptional Structural Compliance of the B₁₂F₁₂^2– Superweak Anion

The single-crystal X-ray structures, thermogravimetric analyses, and/or FTIR spectra of a series of salts of the B₁₂F₁₂^2– anion and homoleptic Ag­(L)_<i>n</i>⁺ cations are reported (L = CH₂Cl₂, <i>n</i> = 2; L = PhCH₃, <i>n</i> = 3; L = CH₃CN; <i>n</i> = 2–4; L = CO, <i>n</i> = 1, 2). The superweak-anion nature of B₁₂F₁₂^2– (Y^2–) was demonstrated by the rapid reaction of microcrystalline Ag₂(Y) with 1 atm of CO to form a nonclassical silver­(I) carbonyl compound with an FTIR ν­(CO) band at 2198 cm^–1 (and with the proposed formula [Ag­(CO)_<i>n</i>]₂[Y]). In contrast, microcrystalline Ag₂(B₁₂Cl₁₂) did not exhibit ν­(CO) bands and therefore did not form Ag­(CO)⁺ species, even after 32 h under 24 atm of CO. When Ag₂(Y) was treated with carbon monoxide pressures higher than 1 atm, a new ν­(CO) band at 2190 cm^–1 appeared, which is characteristic of a Ag­(CO)₂⁺ dicarbonyl cation. Both Ag₂(CH₃CN)₈(Y) and Ag₂(CH₃CN)₅(Y) rapidly lost coordinated CH₃CN at 25 °C to form Ag₂(CH₃CN)₄(Y), which formed solvent-free Ag₂(Y) only after heating above 100 °C. Similarly, Ag₂(PhCH₃)₆(Y) rapidly lost coordinated PhCH₃ at 25 °C to form Ag₂(PhCH₃)₂(Y), which formed Ag₂(Y) after heating above 150 °C, and Ag₂(CH₂Cl₂)₄(Y) rapidly lost three of the four coordinated CH₂Cl₂ ligands between 25 and 100 °C and formed Ag₂(Y) when it was heated above 200 °C. Solvent-free Ag₂(Y) was stable until it was heated above 380 °C. The rapid evaporative loss of coordinated ligands at 25 °C from nonporous crystalline solids requires equally rapid structural reorganization of the lattice and is one of three manifestations of the structural compliance of the Y^2– anion reported in this work. The second, more quantitative, manifestation is that Ag⁺ bond-valence sums for Ag₂(CH₃CN)_<i>n</i>(Y) are virtually constant, 1.20 ± 0.03, for <i>n</i> = 8, 5, 4, because the Y^2– anion precisely compensated for the lost CH₃CN ligands by readily forming the necessary number of weak Ag–F­(B) bonds. The third, and most exceptional, manifestation is that the idealized structural reorganization accompanying the conceptual transformations Ag₂(CH₃CN)₈(Y) → Ag₂(CH₃CN)₅(Y) → Ag₂(CH₃CN)₄(Y) involve close-packed layers of Y^2– anions that sandwich the Ag­(CH₃CN)₄⁺ complexes splitting into staggered flat ribbons of interconnected (Y^2–)₃ triangles that surround the Ag₂(CH₃CN)₅²⁺ complexes on four sides, conceptually re-forming close-packed layers of anions that sandwich the Ag­(CH₃CN)₂⁺ complexes. The interconnected (Y^2–)₃ triangle lattice of anions in Ag₂(CH₃CN)₅(Y) may be the first example of this structure type

Table_1_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Table_3_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Image_1_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.pdf

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Table_6_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Table_11_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Table_12_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Table_7_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Table_5_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p

Table_2_Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.docx

<p>In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3–24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential “off-target” sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.</p