[19–24]). Amplicons were cleaned by binding to AmPure XP paramagnetic beads (Beckman Coulter, Beverly, MA) at DNA:Beads (v/v) ratio of 1:1.8. One of them, IE-mTyrsgRNA, targets the exonic sequence in Tyr exon1, and the other, SDE-mTyrsgRNA, targets the exon1-intron1-2 junction. NGS analysis of allelic variants induced in microinjected mouse blastocysts. In fact, pioneering genetic studies indicated that many of the thalassemia mutations in the β-globin gene affect splice sites and give rise to aberrant splicing patterns[17,18]. Present address: Xiao-Bing Zhang, PhD. Selection with either puromycin or zeocin increased KO efficiency to 95–96%, while double selection with both puromycin and zeocin led to 100% KO (Figure 6F). However, all results reported so far have been obtained by a single set of injections (i.e. [26,48]). The plate was placed in the Biostation Cell Culture Observation System (Nikon) at the University of California Riverside (UCR) Stem Cell Core. pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana. Taken together, these data demonstrate that overexpressing BCL-XL is a universal approach for increasing editing efficiency in human PSCs. Although a reduction of hatch rate was observed in all injected eggs, differences in hatch rates between mock injections and RNP injected eggs were not significant, indicating that RNP complexes had no deleterious effects on embryo viability. To further increase reproducibility of our studies, we carried out these experiments using six different iPSC lines. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, https://doi.org/10.1371/journal.pone.0197567. Only one of six SDE-hATMsgRNA-edited clones expressed ATM, while ATM expression could not be detected in the other five clones. Yu J., Vodyanik M.A., Smuga-Otto K., Antosiewicz-Bourget J., Frane J.L., Tian S., Nie J., Jonsdottir G.A., Ruotti V., Stewart R.et al. These mutations can generate knockout alleles when CRISPR/Cas9 is directed at coding sequences, but due to the variable size of NHEJ-induced indels, generating a full KO in one step cannot always be achieved at high frequency. Furthermore, it was observed that greater than 6 μM concentrations of ribonucleoprotein complexes tend to clog the injection needles quickly during injections, reducing reproducibility and rendering the process much more difficult. Generally 1 μg of Cas9 plasmid, 0.5 μg of sgRNA plasmid, 0.5 μg of sgDocut plasmid (for cutting pDonor in some experiments), 1 μg of pDonor plasmid, and 0.5 μg of BCL2 family gene plasmids were used. Salmon (49) was used to determine transcripts per million (TPM) by aligning the data with the human transcriptome (Gencode Release 19, GRCh37.p13). 2015 Jul;89(7):1023-34. doi: 10.1007/s00204-015-1504-y. Simple microinjection [] or even electroporation [] of the CRISPR/Cas9 system can produce knockout and/or knockin mice easily and efficiently, instead of the time- and labor-consuming gene targeting of embryonic stem cells followed by chimeric mice generation. Yao X., Wang X., Hu X., Liu Z., Liu J., Zhou H., Shen X., Wei Y., Huang Z., Ying W.et al. Drosophila melanogaster and Tribolium castaenum) alters wild type eye color to produce a vermillion phenotype. Data curation,
Again, the experiments were not replicated and statistical analyses were not provided. In the absence of a template, the NHEJ pathway is utilized, introducing variable insertions or deletions (indels) at the DSB site, which may disrupt the open reading frame of the gene and generate a knockout (KO) allele. Although J. coenia mutation rates were lower than that of V. cardui in both experiments, it is not known whether high mutation rates could be achieved by using different concentrations of reagents because only one combination of gRNA and Cas9 protein concentrations was reported.
Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. Overexpression of BCL2 or BCL-XL protects dissociation-induced cell death (35,36). Eye color of adults was recorded and any insects that had a color different from the wild type eye color were genotyped by sequencing amplicons. Both systems have recently been used to create knock-out alleles with great efficiency, and … The National Center for Biotechnology Information (NCBI) peptide database was also searched using a putative H. zea TO peptide sequence translated from the cDNA sequence to identify other insect TO sequences for use in alignments and phylogenetic tree construction.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (. Front Mol Biosci. For clarity, we only showed the top 10 possibilities of on-target cleavage in representative samples. IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain, Mouse ESCs predominantly use the HDR pathway to repair dsDNA damage, relative to somatic cells (81). All groups followed the same cell preparation and electroporation parameters. | It has puzzled us that BCL-XL increases reprogramming efficiency by ∼3-fold in a lentiviral transduction system, while increasing efficiency ∼10-fold in the electroporation system (37). Plant Cell Physiol. Cell survival was determined by cell count on day 1 after electroporation. Investigation, (C) The quantification of annexin V expression in K562-edited cells with SDE and IE hABL-1 sgRNAs showed a higher level of expression in SDE-hABL-1sgRNA edited cells (568,2 mfi) than in IE-hABL-1sgRNA-edited cells (475.5 mfi). https://doi.org/10.1371/journal.pone.0216674.s008, https://doi.org/10.1371/journal.pone.0216674.s009, https://doi.org/10.1371/journal.pone.0216674.s010, https://doi.org/10.1371/journal.pone.0216674.s011, https://doi.org/10.1371/journal.pone.0216674.s012. here. Even when ABT-263 was used to further enrich edited iPSCs, off-target cleavage was undetectable at both sites (Supplementary Table S5A and B). Manipulating factors related to cell death may have unexpected effects on affecting genome editing efficiency in different types of cells. CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. (C–E) Indels (HDR + NHEJ), HDR efficiency, and HDR percentage in all edited cells at AAVS1 (C), CD326 (D), and GAPDH (E) using ssODN donors (single-stranded oligo DNAs) in iPSCs. However, several mutated cell clones (5/6) edited with SDE-hATMsgRNA had no levels of ATM protein that could be detected by WB (Fig 5B). The sgRNA sequences were designed with the web tool of the Spanish National Biotechnology Centre (CNB)-CSIC (http://bioinfogp.cnb.csic.es/tools/breakingcas/). If confirmed we envision that, together with the double cut donor design (17), the success rates of creating compound animal models can be considerably increased (15,87). When the condensation disappeared one hour later, one spot was chosen from each well for time-lapse imaging. Starting 2 hours after electroporation, many cells released multiple small apoptotic bodies indicating apoptosis, whereas a few cells lost viability with the remaining corpse, suggestive of necrosis. By contrast, when we used a Tyr SDE-sgRNA, we detected albino or mosaic mice featuring one allele with a frameshift mutation and another with a mutation but a destroyed splice-donor site. This tool returns results including total read number, HDR read number and percentage, and indels reads (the sum of HDR and NHEJ).
doi: 10.1093/nar/gkaa195. Black and gray circles correspond to null and functional alleles, respectively, while the background indicates the type of mutation (dark blue: splice donor site in-frame and/or frameshift; light blue: frameshift). As such, we decided to edit AAVS1, CD326, and GAPDH loci using ssODNs. Log R ratio (LRR) and B allele frequency (BAF) were used to detect copy number variants (CNVs). Together these data consolidate the conclusion that addition of BCL-XL promotes the survival of plasmid-transfected iPSCs, abrogating the negative selection against high-copy cells. In particular, human PSCs undergo more rapid death after DNA damage-mediated activation of TP53 signaling than differentiated cells (82). To further validate these results, we designed pDonors to insert a short fragment of 6–12 bp at EEF1A1 and GAPDH, allowing assessment of both NHEJ and HDR editing by deep sequencing. As expected, HDR KI efficiency at CTNNB1 increased from 17 to 95% after puromycin selection (Figure 6B). Martins-Taylor K., Nisler B.S., Taapken S.M., Compton T., Crandall L., Montgomery K.D., Lalande M., Xu R.H. Tichy E.D., Pillai R., Deng L., Liang L., Tischfield J., Schwemberger S.J., Babcock G.F., Stambrook P.J. We used FLASH (57) to merge double-ended data and split merged data with the Barcode Splitter tool on Galaxy. No, Is the Subject Area "Mammalian genomics" applicable to this article? The BCL2 family is the central regulator that arbitrates the life-and-death decision. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. These results are consistent with the well-established function of BCL-XL as a potent anti-apoptotic factor. Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/.
Zou J., Maeder M.L., Mali P., Pruett-Miller S.M., Thibodeau-Beganny S., Chou B.K., Chen G., Ye Z., Park I.H., Daley G.Q.et al. Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. © The Author(s) 2018. Deep sequencing showed ∼20% indel efficiency at the PRDM14 on-target locus and ∼30% indel efficiency at CTNNB1 (Supplementary Table S4A and B). (A and B) Effects of BCL2 and MCL1 on human iPSC survival and editing. In addition, RNA transcription from plasmids is highly dependent on the promoter sequence; a promoter from one species may not work optimally in a different species. Human iPSCs were cultured at 37°C with 5% CO2 and were fed with fresh medium every day. For more information about PLOS Subject Areas, click Writing – original draft, Roles Therefore, any delay in transcription of gRNA and Cas from plasmid constructs or translation of Cas mRNA may lead to reduced germline editing efficiency as well as multiple “chimeric” mutations in the embryonic cell population (e.g. A lower level of ATM expression was observed in IE-hATMsgRNA-edited cells, and an even lower level was noted in SDE-hATMsgRNA-edited cells. BCL inhibition further increases editing efficiency in BCL-XL transfected iPSCs. IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain, Roles However, for several reasons, these mutations do not produce the desired null result in all cases, giving rise to a similar but functionally active protein. iPSCs treated in different conditions were harvested three days after electroporation for genomic DNA extraction using the Gentra Puregene Blood Kit (Qiagen) with RNase digestion.
In addition, copy numbers were maintained at ∼400 up until 24 hours after transfection, which can be explained by TP53 activation-induced cell cycle arrest (see the ‘RNA-seq analysis’ section). HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene.
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