RNA-guided AsCas12a- and SpCas9-catalyzed knockout and homology directed repair of the omega-1 locus of the human blood fluke, Schistosoma mansoni

We compared the efficiency and precision of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with SpCas9 of Streptococcus pyogenes aiming to advance functional genomics tools for Schistosoma mansoni. Programmed double stranded cleavage catalyzed by AsCas12a results in a staggered strand break whereas SpCas9 produces a blunt ended chromosomal break. The TTTV, the optimal protospacer adjacent motif for AsCas12a is expected frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions (gRNA:SpCas9:DNA donor ratio and electroporation condition) to edit the ω1 gene. SpCas9 delivered higher efficiency to mutate ω1 target compared to AsCas12a for non-homology end joining (NHEJ)-catalyzed repair (14.04% vs. 10.88%, n = 7 replicates). Most mutations were deletions; SpCas9 induced -3 nt size deletions whereas AsCas12a induced deletions ranging in size from -25 to -2 nt. Although these were less absolute percentage AsCas12a than SpCas9 programmed mutations, the phenotypic outcomes on levels of ω1 transcripts and expressed omega-1 protein were similar. Gene editing efficiency of SpCas9 and AsCas12a markedly increased in the presence of short single stranded donor templates bearing symmetrical homolog arms of 50 nt in length. With AsCas12a, both non-CRISPR target (NT) and target (T) strands of the ω1 gene were tested as homology direct repair (HDR) donor templates. There were 15.67%, 28.71% and 21.43% of NHEJ from 7 pooled genomic DNA from KI_SpCas9, KI_AsCas12a-NT-ssODN and KI_AsCas12a-T-ssODN experiments, respectively. Programmed SpCas9 cleavage led to higher levels than AsCas12a of precise HDR mediated; 17.07%, KI_SpCas9 vs. 14.58%, KI_AsCas12a-NT-ssODN and 12.35%, KI_AsCas12a-T-ssODN (P < 0.0.5), although no significant differences in reduction in ω1 transcripts or of protein levels were apparent. These findings revealed that both AsCas12a and SpCas9 can provide programmed knockout and transgene insertion into genes expressed in the schistosome egg.

Download Full-text

Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

Plant and Cell Physiology ◽

10.1093/pcp/pcab034 ◽

2021 ◽

Author(s):

Daisuke Miki ◽

Rui Wang ◽

Jing Li ◽

Dali Kong ◽

Lei Zhang ◽

...

Keyword(s):

Gene Targeting ◽

Agronomic Traits ◽

Higher Plants ◽

Crop Improvement ◽

Strand Break ◽

End Joining ◽

Homology Directed Repair ◽

Target Dna ◽

Targeted Gene Editing ◽

Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

Download Full-text

Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

10.1101/358424 ◽

2018 ◽

Cited By ~ 2

Author(s):

Wannaporn Ittiprasert ◽

Victoria H. Mann ◽

Shannon E. Karinshak ◽

Avril Coghlan ◽

Gabriel Rinaldi ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Genome Editing ◽

Human Macrophage ◽

Wild Type ◽

End Joining ◽

Chromosomal Breakage ◽

Knock Out ◽

Guide Rna ◽

Homology Directed Repair ◽

Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

Download Full-text

Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1400236111 ◽

2014 ◽

Vol 111 (10) ◽

pp. E924-E932 ◽

Cited By ~ 113

Author(s):

Luther Davis ◽

Nancy Maizels

Keyword(s):

Genome Engineering ◽

Alternative Pathway ◽

Strand Break ◽

End Joining ◽

Single Strand Break ◽

Homology Directed Repair ◽

Single Strand Break Repair ◽

Efficient Alternative ◽

Break Repair ◽

Dna Nicks

DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9D10A nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

Download Full-text

Mapping the Genetic Landscape of DNA Double-strand Break Repair

10.1101/2021.06.14.448344 ◽

2021 ◽

Author(s):

Jeffrey A Hussmann ◽

Jia Ling ◽

Purnima Ravisankar ◽

Jun Yan ◽

Ann Cirincione ◽

...

Keyword(s):

High Throughput Screening ◽

Strand Break ◽

Dna Lesions ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Homology Directed Repair ◽

Genetic Perturbations ◽

Genetic Landscape ◽

Repair Mechanisms

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways that are critical for maintaining genomic integrity. Here we present Repair-seq, a high-throughput screening approach that measures the effects of thousands of genetic perturbations on the distribution of mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair outcomes induced by two programmable nucleases (Cas9 and Cas12a) after knockdown of 476 genes involved in DSB repair or associated processes in the presence or absence of oligonucleotides for homology-directed repair (HDR). The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. Systematic interrogation of these dependencies then uncovered unexpected relationships among DSB repair genes and isolated incompletely characterized repair mechanisms. This work provides a foundation for understanding the complex pathways of DSB repair and for optimizing genome editing across modalities.

Download Full-text

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

International Journal of Molecular Sciences ◽

10.3390/ijms23020631 ◽

2022 ◽

Vol 23 (2) ◽

pp. 631

Author(s):

Wannaporn Ittiprasert ◽

Chawalit Chatupheeraphat ◽

Victoria H. Mann ◽

Wenhui Li ◽

André Miller ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Streptococcus Pyogenes ◽

Human Blood ◽

Gene Knockout ◽

Strand Breaks ◽

Homology Directed Repair ◽

Guide Rnas ◽

Optimized Conditions ◽

Transgene Insertion

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Download Full-text

A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

10.1101/2020.05.11.088252 ◽

2020 ◽

Author(s):

Eric Danner ◽

Mikhail Lebedin ◽

Kathrin de la Rosa ◽

Ralf Kühn

Keyword(s):

Basic Research ◽

Strand Break ◽

End Joining ◽

Linear Amplification ◽

Promising Alternative ◽

Independent Sequence ◽

Replacement Strategy ◽

Homology Directed Repair ◽

Dividing Cells ◽

Non Homologous End Joining

AbstractPrecision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

Download Full-text

Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014176117 ◽

2020 ◽

Vol 117 (44) ◽

pp. 27566-27577

Author(s):

Jacob V. Layer ◽

Lydie Debaize ◽

Alexandria Van Scoyk ◽

Nealia C. House ◽

Alexander J. Brown ◽

...

Keyword(s):

Chromosomal Rearrangements ◽

Strand Break ◽

Double Strand Break ◽

Human Cells ◽

Nonhomologous End Joining ◽

End Joining ◽

Homology Directed Repair ◽

Accessory Subunit ◽

Radiation Induced ◽

Translocation Frequency

Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

Download Full-text

Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

BMC Biology ◽

10.1186/s12915-019-0711-z ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 3

Author(s):

Jes Alexander ◽

Gregory M. Findlay ◽

Martin Kircher ◽

Jay Shendure

Keyword(s):

Genome Editing ◽

Cpg Island ◽

Strand Break ◽

End Joining ◽

Exogenous Dna ◽

Cpg Dinucleotides ◽

Epigenome Editing ◽

Homology Directed Repair ◽

Non Homologous End Joining

Abstract Background Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. Results In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. Conclusions This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

Download Full-text

RNF8 has both KU-dependent and independent roles in chromosomal break repair

Nucleic Acids Research ◽

10.1093/nar/gkaa380 ◽

2020 ◽

Vol 48 (11) ◽

pp. 6032-6052 ◽

Cited By ~ 2

Author(s):

Linda Jillianne Tsai ◽

Felicia Wednesday Lopezcolorado ◽

Ragini Bhargava ◽

Carlos Mendez-Dorantes ◽

Eva Jahanshir ◽

...

Keyword(s):

Double Strand Breaks ◽

End Joining ◽

Protection Factor ◽

Strand Breaks ◽

Homology Directed Repair ◽

Single Strand Annealing ◽

Non Homologous End Joining ◽

Chromosomal Break ◽

Strand Annealing ◽

End Resection

Abstract Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.

Download Full-text

The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

Frontiers in Genetics ◽

10.3389/fgene.2021.823943 ◽

2022 ◽

Vol 12 ◽

Author(s):

Alice Libri ◽

Timea Marton ◽

Ludovic Deriano

Keyword(s):

Dna Repair ◽

Gene Diversity ◽

Receptor Gene ◽

Strand Break ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

End Joining ◽

Homology Directed Repair ◽

Pathway Choice ◽

Non Homologous End Joining

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

Download Full-text