Journal: Genes & Development

Article Title: Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

doi: 10.1101/gad.311084.117

Figure Lengend Snippet: The effect of DSB/repeat distance on the frequency of RMD rearrangements. ( A ) Diagram of the RMD-GFP reporter. Two tandem repeat (R) sequences are separated by 0.4 Mb and positioned such that an RMD generates a Cdkn1a -GFP fusion gene. The RMD is induced by two DSBs between the repeats: one DSB downstream from the 5′ repeat (5′268) and a second DSB at various distances upstream of the 3′ repeat. Shown are the single-guide RNA (sgRNA)/Cas9 targeting sites for each DSB. ( B ) Each sgRNA/Cas9-mediated DSB induces mutagenic end joining at a similar frequency. Shown are representative surveyor assays from wild-type RMD-GFP mouse embryonic stem cells (mESCs) that were untransfected (UT) or transfected with expression plasmids for Cas9 and each sgRNA shown in A . (Arrows) Cleaved products; (*) nonspecific band. Also shown is the frequency of mutagenic end joining using the surveyor nuclease assay, normalized to transfection efficiency, for each sgRNA/Cas9-mediated DSB. n = 6. Error bars indicate SD. P > 0.05, comparing all frequencies using one-way ANOVA with Tukey's test. ( C ) Induction of RMDs using pairs of sgRNAs with Cas9. Shown are the frequencies of GFP + cells normalized to transfection efficiency for RMD-GFP wild-type mESCs transfected with expression plasmids for Cas9 and the sgRNAs shown in A . (Black) n = 8 for single sgRNAs; (blue) n = 6 for sgRNA pairs. Error bars indicate SD. ( D ) GFP + cells harbor the predicted fusion gene. Shown are PCR amplification products from untransfected and sorted GFP + cells from the sgRNA/Cas9 pairs as in C using primers in Cdkn1A (P1) and GFP (P2). Amplification of a fragment from 53bp1 was used as a control. ( E ) Chromosomal DSB/repeat distance has a biphasic influence on the frequency of RMDs in wild-type mESCs. Shown are the data from the sgRNA pairs as in C but plotted as the percentage of GFP + cells versus the distance (in base pairs) between the 3′ chromosomal DSB and the 3′ repeat. n = 6. Error bars indicate SD. (ns) Not significant; (*) P ≤ 0.0018 using one-way ANOVA with Tukey's test.

Article Snippet: Each sgRNA sequence is shown in Supplemental Table S1 and was expressed from the px330 plasmid, which coexpresses Cas9 (Addgene, 42230; generously deposited by Dr. Feng Zhang) ( ).

Techniques: Transfection, Expressing, Nuclease Assay, Amplification, Control

Journal: Genes & Development

Article Title: Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

doi: 10.1101/gad.311084.117

Figure Lengend Snippet: RMDs are suppressed by RAD51 when the 3′ DSB is ≥3.3 kb. ( A ) Two dominant-negative inhibitors of RAD51—3xFlag-NLS-BRC3 (3xfNLS-BRC3) and RAD51-K133R—disrupt HDR. Wild-type mESCs with DR-GFP were transfected with expression plasmids for an sgRNA targeting this reporter (gDR) and Cas9 along with expression plasmids for 3xfNLS-BRC3, RAD51-K133R, or a control EV. Shown are GFP + frequencies normalized to transfection efficiency. n = 6. Error bars indicate SD. (*) P = 0.0001 compared with EV using one-way ANOVA with Dunnett's test. ( B ) Expression of 3xfNLS-BRC3 impairs RAD51 IRIF formation. Shown are representative images of DAPI, Flag, and RAD51 staining in wild-type mESCs transfected with 3xfNLS-BRC3 and exposed to 10 Gy IR followed by 6 h of recovery. Bar, 10 µm. Also shown is the number of RAD51 IRIFs for individual Flag − and Flag + cells. n = 70. Lines represent the mean with SD. (*) P < 0.0001 using an unpaired two-tailed t -test. ( C ) The effect of 3xfNLS-BRC3 and RAD51-K133R on RMD frequency. Shown are the frequencies of GFP + cells normalized to transfection efficiency for RMD-GFP in wild-type mESCs transfected with expression plasmids for a series of sgRNA pairs and Cas9 along with expression plasmids for 3xfNLS-BRC3, RAD51-K133R, or control EV. Also shown are the data plotted as percentage of GFP + versus 3′ DSB/repeat distance as in E. n = 6. Error bars indicate SD. (*) P ≤ 0.0017; (†) P = 0.0260, each compared with EV using one-way ANOVA with Dunnett's test.

Article Snippet: Each sgRNA sequence is shown in Supplemental Table S1 and was expressed from the px330 plasmid, which coexpresses Cas9 (Addgene, 42230; generously deposited by Dr. Feng Zhang) ( ).

Techniques: Dominant Negative Mutation, Transfection, Expressing, Control, Staining, Two Tailed Test

Journal: Genes & Development

Article Title: Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

doi: 10.1101/gad.311084.117

Figure Lengend Snippet: RAD52, CtIP, and BRCA1 promote RMDs. ( A ) RAD52 promotes RMDs for 3′ DSB/repeat distances of ≥3.3 kb. Wild-type or Rad52 −/− mESCs with RMD-GFP were transfected with the respective sgRNA/Cas9 expression vectors along with a control EV or V5-RAD52 complementation vector. Shown is the percentage of GFP + from these experiments, normalized to transfection efficiency. n = 6. Error bars indicate SD. (*) P ≤ 0.0009 distinct from wild-type EV; (‡) P ≤ 0.0025 for Rad52 −/− mESCs EV versus V5-RAD52, both using one-way ANOVA with Tukey's test. Wild-type frequencies are the same as in C. Also shown is RT–PCR analysis of the RAD52 transcript in wild-type and Rad52 −/− mESCs using RNA either treated with reverse transcriptase (RT) or mock-treated as well as immunoblotting analysis confirming RAD52 expression using the V5 immunotag. (*) Nonspecific band. ( B ) Depletion of BRCA1 or CtIP causes a reduction in HDR and RMDs but not end joining (distal EJ). Wild-type mESCs with various reporters were transfected with the respective sgRNA/Cas9 expression plasmids along with a nontargeting siRNA (siCTRL), a pool of four BRCA1 siRNAs (siBRCA1), or a pool of four CtIP siRNAs (siCtIP). Shown is the percentage of GFP + from these experiments, normalized to transfection efficiency. n = 6 for DR-GFP and EJ5-GFP; n = 9 for RMD-GFP. Error bars indicate SD. (*) P = 0.0001 versus siCTRL using one-way ANOVA with Dunnett's test. Also shown is immunoblotting analysis confirming depletion of BRCA1 and CtIP by the respective siRNAs.

Article Snippet: Each sgRNA sequence is shown in Supplemental Table S1 and was expressed from the px330 plasmid, which coexpresses Cas9 (Addgene, 42230; generously deposited by Dr. Feng Zhang) ( ).

Techniques: Transfection, Expressing, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Western Blot

Journal: Genes & Development

Article Title: Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

doi: 10.1101/gad.311084.117

Figure Lengend Snippet: KU70 and XRCC4 suppress RMDs, whereas 53BP1 does not have a substantial influence. ( A ) KU70 suppresses RMDs. Wild-type or Ku70 −/− mESCs with RMD-GFP were transfected with the respective sgRNA/Cas9 expression vectors along with control EV or KU70 complementation vector. Shown are the percentages of GFP + frequencies from these experiments, normalized to transfection efficiency. Also shown are immunoblots confirming KU70 expression. n = 6 for wild-type EV; n = 9 for Ku70 −/− EV and KU70 complemented. Error bars indicate SD. (*) P = 0.0001; (†) P = 0.0077, for Ku70 −/− EV versus wild-type EV or for Ku70 −/− EV versus KU70 complemented using one-way ANOVA with Dunnett's test. ( B ) XRCC4 suppresses RMDs. Xrcc4 −/− mESCs with RMD-GFP were transfected with the respective sgRNA/Cas9 expression vectors along with a control EV or an expression vector for human XRCC4 (hXRCC4). Shown are the frequencies of the percentages of GFP + cells from these experiments, normalized to transfection efficiency. The wild-type frequencies from A are included for comparison. n = 6. Error bars indicate SD. (*) P ≤ 0.0003; (†) P ≤ 0.0313, for Xrcc4 −/− EV versus wild type or for Xrcc4 −/− EV versus hXRCC4 using one-way ANOVA with Dunnett's test. Also shown is an immunoblot confirming expression of hXRCC4. Wild-type mESC immunoblotting signals are not shown, since this antibody does not recognize mouse XRCC4. ( C ) 53BP1 does not have an obvious effect on the frequency of RMDs. The mESC lines wild type, 53bp1 −/− , Ku70 −/− , and Ku70 −/− 53bp1 −/− , each with RMD-GFP, were transfected with the respective sgRNAs/Cas9 plasmids, and nontargeting siCTRL was also included. Shown are the percentages of GFP + cells from these experiments, normalized to transfection efficiency. Wild-type frequencies are the same as in B. n = 9. Error bars indicate SD. (*) P ≤ 0.0021; (†) P = 0.0389, comparisons versus wild-type using one-way ANOVA with Dunnett's test.

Article Snippet: Each sgRNA sequence is shown in Supplemental Table S1 and was expressed from the px330 plasmid, which coexpresses Cas9 (Addgene, 42230; generously deposited by Dr. Feng Zhang) ( ).

Techniques: Transfection, Expressing, Control, Plasmid Preparation, Western Blot, Comparison

Journal: Genes & Development

Article Title: Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

doi: 10.1101/gad.311084.117

Figure Lengend Snippet: Loss of KU70 and 53BP1 affects the requirement for BRCA1 and CtIP for RMDs. ( A ) Depletion of BRCA1 fails to reduce RMD frequency in cells deficient in KU70 and/or 53BP1 and indeed causes an increase in KU70-deficient cells. Cell lines shown in C were transfected with the respective sgRNA/Cas9 expression plasmids along with either siCTRL or siBRCA1. Shown are the percentages of GFP + cells from these experiments, normalized to transfection efficiency. The frequencies for wild type and each cell line with siCTRL are the same as C. n = 9. Error bars indicate SD. (*) P ≤ 0.0003 for siCTRL versus siBRCA1 for all cell lines at each sgRNA pair using an unpaired t -test with the Holm-Sidak correction. Also shown is immunoblotting to confirm depletion of BRCA1 in each cell line. The wild-type immunoblot is the same as B and is shown for comparison. ( B ) Depletion of CtIP shows diminished effects on RMDs in cells deficient in KU70 and/or 53BP1 compared with wild type. Cell lines were transfected as in A but including siCtIP. Shown are the frequencies of GFP + cells normalized to transfection efficiency for these experiments. As in A , the frequencies for wild type and each cell line with siCTRL are the same as C; namely, the data shown are from experiments with siCTRL, siBRCA1, and siCtIP tested in parallel for each cell line. n = 9. Error bars indicate SD. (*) P ≤ 0.003; (†) P < 0.03 for siCTRL versus siCtIP for all cell lines at each sgRNA pair using an unpaired t -test with the Holm-Sidak correction. Also shown is immunoblotting to confirm depletion of CtIP in each cell line. The wild-type immunoblot is the same as B and is shown for comparison.

Article Snippet: Each sgRNA sequence is shown in Supplemental Table S1 and was expressed from the px330 plasmid, which coexpresses Cas9 (Addgene, 42230; generously deposited by Dr. Feng Zhang) ( ).

Techniques: Transfection, Expressing, Western Blot, Comparison

Journal: Genes & Development

Article Title: Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

doi: 10.1101/gad.311084.117

Figure Lengend Snippet: Introducing sequence divergence between the repeats substantially suppresses RMD in a manner dependent on the mismatch repair factor MSH2. ( A ) Introducing sequence divergence between the repeats markedly suppresses RMD frequency at multiple DSB/repeat distances. Shown are variants of the RMD-GFP reporter: 1% RMD-GFP and 3% RMD-GFP, with three and eight mutations, respectively, which are equally dispersed in the 3′ repeat (R) fused to GFP and do not affect the respective codon in the repeat (i.e., are silent mutations). Each reporter was integrated into wild-type mESCs and transfected with the respective sgRNA/Cas9 pairs shown in A. Shown is the frequency of GFP + cells normalized to transfection efficiency for these experiments. n = 15 for RMD-GFP. n = 6 from A, with additional replicates performed later with the divergent reporter cell lines; n = 9 for 1% RMD-GFP and 3% RMD-GFP. Error bars indicate SD. (*) P = 0.0001 comparisons versus RMD-GFP using one-way ANOVA with Dunnett's test. ( B ) Loss of MSH2 increases the frequency of RMDs between divergent repeats. Msh2 −/− mESCs with the 1% and 3% reporters shown in A were transfected with the respective sgRNA/Cas9 expression vectors along with a control EV or MSH2 complementation vector. Shown are the percentages of GFP + frequencies normalized to transfection efficiency along with the wild-type frequencies from A . n = 9. Error bars indicate SD. (*) P ≤ 0.001 comparisons versus Msh2 −/− EV using one-way ANOVA with Dunnett's test. Also shown is immunoblot analysis confirming MSH2 expression.

Article Snippet: Each sgRNA sequence is shown in Supplemental Table S1 and was expressed from the px330 plasmid, which coexpresses Cas9 (Addgene, 42230; generously deposited by Dr. Feng Zhang) ( ).

Techniques: Sequencing, Transfection, Expressing, Control, Plasmid Preparation, Western Blot

Journal: Genes & Development

Article Title: Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements

doi: 10.1101/gad.311084.117

Figure Lengend Snippet: MSH2 has a dominant role compared with KU70 to suppress RMDs between divergent repeats. ( A ) KU70 suppresses RMDs between divergent repeats. Ku70 −/− mESCs with the 1% RMD-GFP and 3% RMD-GFP reporters were transfected as in A. Shown are the percentages of GFP + cells from these experiments, normalized to transfection efficiency, with the wild-type frequencies from A shown for comparison. n = 9. Error bars indicate SD. (*) P ≤ 0.0085; (†) P ≤ 0.0432 for Ku70 −/− EV versus wild type or for Ku70 −/− EV versus KU70 expression vector using one-way ANOVA with Dunnett's test. ( B ) Loss of both MSH2 and KU70 causes a marked increase in RMDs between divergent repeats compared with the single mutants, and MSH2 loss has a greater effect than KU70 loss. Msh2 −/− Ku70 −/− mESCs with the 1% RMD-GFP and 3% RMD-GFP reporters were transfected with the respective sgRNA/Cas9 expression plasmids along with control EV. Shown are the percentages of GFP + cells from these experiments, normalized to transfection efficiency, with the wild-type, Msh2 −/− , and Ku70 −/− frequencies from and A shown for comparison. n = 9. Error bars indicate SD. (*) P ≤ 0.0003 for Msh2 −/− Ku70 −/− versus every other cell line; (‡) P ≤ 0.0009 for Msh2 −/− versus Ku70 −/− using one-way ANOVA with Tukey's test. ( C ) Expression of KU70 or MSH2 suppresses RMDs between divergent repeats in Msh2 −/− Ku70 −/− mESCs, with MSH2 having a greater effect. Msh2 −/− Ku70 −/− mESCs with the 1% RMD-GFP and 3% RMD-GFP reporters were transfected as in B but including expression vectors for KU70, MSH2, or EV. Shown are the percentages of GFP + cells from these experiments, normalized to transfection efficiency, with wild-type frequencies from A shown for comparison. n = 6. Error bars indicate SD. (*) P ≤ 0.0003 for Msh2 −/− Ku70 −/− EV versus all other conditions; (‡) P ≤ 0.015 for MSH2 versus KU70 complementation in Msh2 −/− Ku70 −/− cells using one-way ANOVA with Tukey's test.

Article Snippet: Each sgRNA sequence is shown in Supplemental Table S1 and was expressed from the px330 plasmid, which coexpresses Cas9 (Addgene, 42230; generously deposited by Dr. Feng Zhang) ( ).

Techniques: Transfection, Comparison, Expressing, Plasmid Preparation, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: LPG2 Gene Duplication in Leishmania infantum : A Case for CRISPR-Cas9 Gene Editing

doi: 10.3389/fcimb.2020.00408

Figure Lengend Snippet: Generation of LPG2 knockout using CRISPR/Cas9. (A) Western blot analysis of L. infantum promastigotes expressing Cas9 (gRNA440 and gRNA516). (B) Growth curves of L. infantum wild-type (WT) and L. infantum -Cas9 (gRNA440 and gRNA516) promastigotes. (C) Agglutination assay using the CA7AE monoclonal antibody and associated growth curves. (D) Agglutination assay using Ricin 120 lectin and associated growth curves, demonstrating the selection of Δ lpg2 . The arrows indicate the time points where antibody (CA7AE) or lectin (Ricin-120) were added to the cultures, exemplifying the typical agglutination results observed.

Article Snippet: The annealed guide sequence was then cloned into the gRNA and Cas9 coexpression vector (pLdCN) (Addgene plasmid #84290; http://n2t.net/addgene:84290 ; RRID:Addgene_84290 ) previously digested with Bbs I.

Techniques: Knock-Out, CRISPR, Western Blot, Expressing, Agglutination, Selection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: LPG2 Gene Duplication in Leishmania infantum : A Case for CRISPR-Cas9 Gene Editing

doi: 10.3389/fcimb.2020.00408

Figure Lengend Snippet: Molecular characterization of Δ lpg2 and reduced virulence phenotype. (A) Chromatogram and translated sequence showing the region of the LPG2 gene in which the precise insertion of a stop codon (denoted by an *) occurred by homologous recombination at the cleavage site of the Cas9 enzyme (nucleotides in red). The oligodonor sequence is underlined and the gRNA440 sequence is highlighted in blue. (B) Western blot analysis of the expression of LPG and PPGs in L. infantum promastigotes WT and Δ lpg2 clones. (C) Confocal immunofluorescence analysis of WT and Δ lpg2 (clone G6) parasites. Late log-phase promastigotes were adhered on Poly-L-Lysine-coated glass coverslips, fixed and incubated with the CA7AE antibody to visualize LPG and other Gal(β1,4)Man(α1-PO4) repeating unit-containing PGs (green). Differential interference contrast (DIC) for Δ lpg2 is shown in the upper left panel. Scale bar: 10 μm. (D) Axenic growth curves of late log-phase promastigotes of L. infantum wild-type (WT) and clone G6 Δ lpg2 , each point represents mean and SE. Data are representative of at least three independent assays and were collected in triplicate for each experimental condition. (E) Area under the curve (AUC) analysis of growth curves presented in (D) , *** p < 0.01. (F) Reduced virulence of Δ lpg2 parasites in human neutrophils. Human neutrophils were infected with L. infantum Ba262 wild-type and Δ lpg2 promastigotes for 3 h. Numbers of viable promastigotes shown after 24 h, with each point on the graph representing the cells from a health donor. Statistical differences were evaluated using the Student t- test, **** p < 0.001.

Article Snippet: The annealed guide sequence was then cloned into the gRNA and Cas9 coexpression vector (pLdCN) (Addgene plasmid #84290; http://n2t.net/addgene:84290 ; RRID:Addgene_84290 ) previously digested with Bbs I.

Techniques: Sequencing, Homologous Recombination, Western Blot, Expressing, Clone Assay, Immunofluorescence, Incubation, Infection

Journal: Nature genetics

Article Title: Alteration of genome folding via contact domain boundary insertion

doi: 10.1038/s41588-020-0680-8

Figure Lengend Snippet: CRISPR dissections of insertion, and RAD21 distribution at C21S2. a, TSS_sgRNA_2&4 and TSS_sgRNA_3&4 (as in Ext Data Fig. 8a) reduce transcription more effectively at C21S2 in CRISPR-Cas9 RNP-transfected cells. N=1 experiment. b, Deletion of the inserted CBS reduces but does not abolish transcription at C21S2. c, Clone 21 TSS- derived from TSS_sgRNA_2&4-edited Clone 21 abrogates transcription, with the CBS intact. d, Clone 21 CTCF-/TSS- #1: derived from CBS-disrupted Clone 21 (b) further edited with TSS_sgRNA_2&4. e, Clone 21 CTCF-/TSS- #2: derived from CBS-disrupted Clone 21 (b) further edited with TSS_sgRNA_3&4. In (b)-(e), N=2 independent experiments for each genotype. In (f)-(h), red arrow: insertion site; green or blue arrow: downstream CBSs; orange arrowhead in the browser tracks: locus/orientation of the insertion. f, g, Hi-C maps of Clone 21 CTCF-/TSS- #1 (d) and of Clone 21 CTCF-/TSS- #2 (e), respectively, at C21S2: deletions of both the CBS and the TSS restore the domain configuration close to pre-insertion level . h, Capture-C and corresponding data tracks showing that CTCF-/TSS- rescues local chromatin contact pattern close to that of WT. Differentially bound RAD21 peaks (R6, R7) upon CBS-TSS insertion highlighted. Directionality Index of Clone 21 CTCF-/TSS- #1 Capture-C: . In (f)-(h), each Hi-C/Capture-C depicts merged data from at least 2 independent experiments for each genotype. 2 CTCF/RAD21 ChIP-seq and 1 H3K27ac ChIP-seq for each genotype, with 1 of each shown. i, Pairwise comparisons between genotypes of RAD21 binding at two RAD21 peaks (R6 and R7, as in h and - ). N on-Clone 21: 3 genotypes without Clone 21 insertions, each with 2 ChIP-seq replicates. All others: 1 genotype, each with 2 ChIP-seq replicates. P-values (not adjusted for multiple comparisons) are derived from a two-sided Wald test through DiffBind.

Article Snippet: Oligos ( ) encoding this gRNA were annealed and cloned into a plasmid co-expressing Cas9 and gRNA, with GFP, modified from pX330 (Addgene, 42230).

Techniques: CRISPR, Transfection, Derivative Assay, Hi-C, Capture-C, ChIP-sequencing, Binding Assay