Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

Lee, Ho Joung; Kim, Hyun Ju; Lee, Sang Jun

doi:10.3390/ijms22126457

Cited by 12 publications

(16 citation statements)

References 32 publications

(42 reference statements)

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“…Moreover, reportedly, the truncation of guide RNA reduces the off-target effect of Cas9 . It was recently reported that 2-nucleotide (nt) truncations at the 5′-end of a single molecular guide RNA (sgRNA) enhanced Cas9-mediated single-base substitution in the microbial genome . As in the case of Cas9, the 3′-end-truncated crRNA-Cpf1 complex can cause double-strand breaks in the target DNA even when 4–6 nt are truncated at the 3′-end of the canonical 23-nt spacer of the CRISPR/Cpf1 crRNA. , In this study, we systematically investigated the relationship between single-base mismatch tolerance and 3′-end nucleotide truncation in the CRISPR/Cpf1 crRNA.…”

Section: Introductionmentioning

confidence: 99%

Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3′-End-Truncated crRNAs

et al. 2022

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Mismatch tolerance, a cause of the off-target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3′-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 in Escherichia coli . Five nucleotides could be maximally truncated at the crRNA 3′-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3′-end-truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3′-end-truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB . Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution.

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Section: Introductionmentioning

confidence: 99%

Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3′-End-Truncated crRNAs

et al. 2022

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“…CpG methylation at the promoters of genes is correlated with repression of those genes, , and dCas9 fused to DNMT3A can de novo methylate CpGs when directed to a promoter of interest and cause gene repression. − In an initial screen in transfection-efficient HEK293FT cells, we directed a dCas9-DNMT3A fusion construct with 20 independent 17 bp truncated gRNAs to the ANTXR2 promoter and the associated CpG island to determine which targeted protospacer would generate the most repression of the gene (Figure A–C). Previous reports indicate that truncated gRNAs (less than the standard 20 bp) have low levels of off-target interactions , and are advantageous because even one mismatch in the target sequence greatly inhibits off-target binding. , Off-target analysis of the 20 designed truncated gRNAs in this study using Cas-OFFinder found that only four of the gRNAs designed have potential single mismatch off-target genomic sites (Table S1), suggesting that the truncated gRNAs used in this study have great potential for specific on-target binding. We found 4 gRNAs from the screen that repressed ANTXR2 48% or more, with gRNA-9 creating the most (70%) repression (Figure D).…”

Section: Resultsmentioning

confidence: 54%

“…Previous reports indicate that truncated gRNAs (less than the standard 20 bp) have low levels of off-target interactions 22,23 and are advantageous because even one mismatch in the target sequence greatly inhibits off-target binding. 22,24 Off-target analysis of the 20 designed truncated gRNAs in this study using Cas-OFFinder 25 found that only four of the gRNAs designed have potential single mismatch off-target genomic sites (Table S1), suggesting that the truncated gRNAs used in this study have great potential for specific on-target binding. We found 4 gRNAs from the screen that repressed ANTXR2 48% or more, with gRNA-9 creating the most (70%) repression (Figure 2D).…”

Section: ■ Results and Discussionmentioning

confidence: 87%

Host Cell Transcriptional Tuning with CRISPR/dCas9 to Mitigate the Effects of Toxin Exposure

et al. 2022

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Anthrax infection is caused byBacillus anthracis, a bacterium that once established within the host releases lethal toxin (LeTx). Anthrax LeTx is internalized by the capillary morphogenesis protein 2/anthrax toxin receptor 2 (CMG2/ANTXR2) cell surface receptor on mammalian cells. Once inside the cell, LeTx cleaves mitogen-activated protein kinases (MAPKs), ultimately leading to cell death. Previous reports have shown that decreased expression of ANTXR2 reduces cell susceptibility to LeTx. By ablating the ANTXR2 gene in cells in vitro, we observed complete resistance to LeTx-induced cell death. Here, we directed CRISPR/dCas9-based tools to the ANTXR2 promoter to modulate ANTXR2 expression without altering the underlying gene sequence in human cell lines that express the receptor at high levels. We hypothesized that downregulating the expression of the ANTXR2 gene at the genomic level would mitigate the impact of toxin exposure. In one epigenetic editing approach, we employed the fusion of DNMT3A DNA methyltransferase and dCas9 (dCas9-DNMT3A) to methylate CpGs within the CpG island of the ANTXR2 promoter and found this repressed ANTXR2 gene expression resulting in significant resistance to LeTx-induced cell death. Furthermore, by multiplexing gRNAs to direct dCas9-DNMT3A to multiple sites in the ANTXR2 promoter, we applied a broader distribution of CpG methylation along the gene promoter resulting in enhanced repression and resistance to LeTx. In parallel, we directed the dCas9-KRAB-MeCP2 transcriptional repressor to the ANTXR2 promoter to quickly and robustly repress ANTXR2 expression. With this approach, in as little as two weeks, we created resistance to LeTx at a similar level to ANTXR2 gene-ablated cells. Overall, we present a transcriptional tuning approach to inhibit the effects of LeTx and provide a framework to repress toxin-binding cell surface receptors.

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“…The sgRNA targets were located close to the N-terminus. Overall, they had only few predicted off-targets, which could be neglected due to their location in intergenic or intronic sequences with a minimum of three to four mismatches [ 34 ]. Finally, both proteins could be addressed due to the accuracy of the CRISPR/Cas9 method [ 35 , 36 ].…”

Section: Discussionmentioning

confidence: 99%

TTC30A and TTC30B Redundancy Protects IFT Complex B Integrity and Its Pivotal Role in Ciliogenesis

Hoffmann

Bolz

Junger

et al. 2022

Genes

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Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia. The dysfunction of IFT leads to ciliopathies of variable severity. Two of the IFT-B components are the paralogue proteins TTC30A and TTC30B. To investigate whether these proteins constitute redundant functions, CRISPR/Cas9 was used to generate single TTC30A or B and double-knockout hTERT-RPE1 cells. Ciliogenesis assays showed the redundancy of both proteins while the polyglutamylation of cilia was affected in single knockouts. The localization of other IFT components was not affected by the depletion of a single paralogue. A loss of both proteins led to a severe ciliogenesis defect, resulting in no cilia formation, which was rescued by TTC30A or B. The redundancy can be explained by the highly similar interaction patterns of the paralogues; both equally interact with the IFT-B machinery. Our study demonstrates that a loss of one TTC30 paralogue can mostly be compensated by the other, thus preventing severe ciliary defects. However, cells assemble shorter cilia, which are potentially limited in their function, especially because of impaired polyglutamylation. A complete loss of both proteins leads to a deficit in IFT complex B integrity followed by disrupted IFT and subsequently no cilia formation.

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Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

Cited by 12 publications

References 32 publications

Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3′-End-Truncated crRNAs

Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3′-End-Truncated crRNAs

Host Cell Transcriptional Tuning with CRISPR/dCas9 to Mitigate the Effects of Toxin Exposure

TTC30A and TTC30B Redundancy Protects IFT Complex B Integrity and Its Pivotal Role in Ciliogenesis

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