CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack. Here, we tested the antiviral activity of seven dual crRNA combinations and analyzed the HIV proviral genomes for mutations at the target sites. We demonstrated that dual crRNA combinations exhibit more robust antiviral activity than a single crRNA attack and, more important, that the dual-crRNA therapy can prevent virus escape in long-term cultures. We confirmed the absence of any replication-competent virus in these apparently cured cultures. Surprisingly, we did not detect excision of the HIV sequences located between two Cas12a cleavage sites. Instead, we observed almost exclusively HIV inactivation by "hypermutation," that is, the introduction of indel mutations at both target sites due to the error-prone cellular DNA repair machinery. 

http://www.cell.com/article/S2329050122000262/pdf

A combinatorial CRISPR-Cas12a attack on HIV DNA

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR-Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations.

Microsatellite expansions are the cause of more than 20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, a new assay was developed to test several CRISPR-Cas nucleases on microsatellites involved in human diseases, by measuring at the same time double-strand break rates, DNA end resection and homologous recombination efficacy. Broad variations in nuclease performances were detected on all repeat tracts. Streptococcus pyogenes Cas9 was the most efficient of all. All repeat tracts did inhibit double-strand break resection. We demonstrate that secondary structure formation on the guide RNA was a major determinant of nuclease efficacy. Using deep sequencing, off-target mutations were assessed genomewide. Out of 221 CAG/CTG or GAA/TTC trinucleotide repeats of the yeast genome, three were identified as carrying statistically significant low frequency mutations, corresponding to off-target effects.

/pdf/differential-efficacies-of-cas-nucleases-on-microsatellites-vc89jx9wtu.pdf

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations

To study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, low off-target efficiency, and the same editing efficiency as Cas9. There are numerous studies on enrichment sequencing using Cas9 combined with nanopore, while there are only a few studies on the enrichment sequencing of long and highly repetitive genes using Cpf1. We developed Cpf1-based enrichment combined with ONT sequencing (CEO) to characterize the B. mori FibH gene, which is composed of many repeat units with a long and GC-rich sequence up to 17 kb and is not easily amplified by means of a polymerase chain reaction (PCR). CEO has four steps: the dephosphorylation of genomic DNA, the Cpf1 targeted cleavage of FibH, adapter ligation, and ONT sequencing. Using CEO, we determined the fine structure of B. moriFibH, which is 16,845 bp long and includes 12 repetitive domains separated by amorphous regions. Except for the difference of three bases in the intron from the reference gene, the other sequences are identical. Surprisingly, many methylated CG sites were found and distributed unevenly on the FibH repeat unit. The CEO we established is an available means to depict highly repetitive genes, but also a supplement to the enrichment method based on Cas9.

Precise Characterization of Bombyx mori Fibroin Heavy Chain Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies.

Trinucleotide repeat expansions are responsible for two dozen human disorders. Contracting expanded repeats by Double-Strand Break Repair (DSBR) might be a therapeutic approach. Given the complexity of manipulating human cells, recent assays were made to quantify DSBR efficacy in yeast, using a fluorescent reporter. In this study DSBR is characterized with an interdisciplinary approach, linking large population dynamics and individual cells. Time-resolved molecular measurements of changes in the population are first confronted to a coupled differential equation model to obtain repair processes rates. Comparisons with measurements in microfluidic devices, where the progeny of 80-150 individual cells are followed, show good agreement between individual trajectories and mathematical and molecular results. Further analysis of individual progenies shows the heterogeneity of individual cell contributions to global repair efficacy. Three different categories of repair are identified: high-efficacy error-free, low-efficacy error-free and low-efficacy error-prone. These categories depend on the type of endonuclease used and on the target sequence.

Single-cell microfluidic analysis unravels individual cellular fates during Double-Strand Break Repair

Cells can repair a double-strand break (DSB) by homologous recombination if a homologous sequence is provided as a template. This can be achieved by classical gene conversion (with or without crossover) or by single-strand annealing (SSA) between two direct repeat sequences flanking the DSB. To initiate SSA, single-stranded regions are needed adjacent to the break, extending up to the direct repeats in such a way that complementary strands can anneal to each other to repair the DSB. In the present protocol, we describe a GFP reporter assay in Saccharomyces cerevisiae allowing for the quantification of nuclease efficacy at inducing a DSB, by monitoring the reconstitution of a functional GFP gene whose expression can be rapidly quantified by flow cytometry.

Bruno Dumas

Papers

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations.

Monitoring Double-Strand Break Repair of Trinucleotide Repeats Using a Yeast Fluorescent Reporter Assay.

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations