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Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression.
CRISPR/Cas9 has been extensively used for gene knockout, enabling functional studies of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, commonly used Cas9 systems usually exhibit limited and variable efficiencies, and identifying single-guide RNAs (sgRNAs) with high cleavage activity-while avoiding ineffective ones-remains a major challenge. To address these issues, we generated a doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line and developed it into an optimized gene knockout system through systematically refining critical parameters. Through this optimization, the system achieved stable INDELs (Insertions and Deletions) efficiencies of 82-93% for single-gene knockouts, over 80% for double-genes knockouts, and up to 37.5% homozygous knockout efficiency for large DNA fragment deletions. Moreover, using this optimized system, we precisely evaluated three widely used gRNA scoring algorithms and integrated Western blotting to rapidly identify the ineffective sgRNA. As a result, among the tested algorithms, Benchling provided the most accurate predictions. Notably, we identified an ineffective sgRNA targeting exon 2 of ACE2, where the edited cell pool exhibited 80% INDELs but retained ACE2 protein expression. Together, these findings provide a robust framework for improving gene knockout efficiency in hPSCs and offer practical guidance for reliable sgRNA selection in gene editing experiments.
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Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9-RecA system.
OBJECTIVE: To develop an inducible CRISPR/Cas9-Recombinase A (RecA) system to manipulate genes in Nonomuraea gerenzanensis effectively. RESULTS: Compared with traditional homologous recombination, the inducible CRISPR/Cas9 system achieved 68.8% editing efficiency, whereas, with both the inducible Cas9 and the overexpressed RecA, the efficiency of the combined genome editing system reached 100%. The dbv23-deleted mutant obtained by the inducible CRISPR/Cas9-RecA system was confirmed to produce more A40926 with an approximate yield of 200 mg L-1 than that of around 150 mg L-1 produced by the wild-type strain. CONCLUSIONS: This inducible CRISPR/Cas9-RecA system was successfully constructed and can be utilized as an efficient genome editing tool for Actinomyces able to shorten editing time simultaneously.
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Double step screening using endogenous marker improves relative gene targeting efficiency in Arabidopsis.
Gene targeting (GT) is a powerful tool for manipulating endogenous genomic sequences as intended. However, its efficiency is rather low, especially in seed plants. Numerous attempts have been made to improve the efficiency of GT via the CRISPR/Cas systems in plants, but these have not been sufficiently effective to be used routinely by everyone. Here, we report a surrogate screening method that improves the relative efficiency of CRISPR/Cas9-mediated GT in Arabidopsis. Our findings indicate that simultaneous mutagenesis of the endogenous MAR1 gene, which results in kanamycin resistance, can be employed to efficiently screen for precise and heritable GT events at multiple endogenous sites in the Arabidopsis genome. In this study, we demonstrate that a double-step screening strategy can achieve up to a four-fold increase in the efficiency of GT in Arabidopsis. The principle of this surrogate system has the potential to be widely applied.
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[Enhancement can do harm].
Inactivation of the CCR5 gene by CRISPR editing in human embryos, as recently attempted in China, was touted as a positive change for the babies involved since it was expected to impart resistance to HIV infection. However, it turns out that the absence of CCR5 is not neutral but actually decreases fitness, as shown by survival analysis of population data in the UK biobank. This underlines the pitfalls of genetic enhancement, and emphasizes that any germline modification must be preceded by in-depth studies to exclude unforeseen negative effects. ‡.
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Genome editing in animals: why FDA regulation matters.
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Parents weigh promise and risks of germline editing.
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Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line.
The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein's catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5-2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.
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Comparison of CRISPR-Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS.
The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR-Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR-Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR-Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.
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CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function.
The CRISPR/Cas9 technology allows fast and marker-less genome engineering that can be employed to study secondary metabolism in actinobacteria. Here, we report a standard experimental protocol for the deletion of a biosynthetic gene in a Streptomyces species, using the vector pCRISPomyces-2 developed by Huimin Zhao and collaborators. We also describe how carrying out metabolite analysis can reveal the putative biosynthetic function of the inactivated gene.
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Genome Editing of Silkworms.
Silkworm is a lepidopteran insect that has been used as a model for a wide variety of biological studies. The microinjection technique is available and it is possible to cause transgenesis as well as target gene disruption via the genome editing technique. TALEN-mediated knock-out is especially effective in this species. We also succeeded in the precise and efficient integration of a donor vector using the Precise Integration into Target Chromosome (PITCh) method. Here, we describe protocols for ZFN, TALEN, and CRISPR/Cas9-mediated genome editing as well as the PITCh technique in the silkworm. We consider that all of these techniques can contribute to the further promotion of various biological studies in the silkworm and other insect species.
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