Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.

Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex.

Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage.

The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications.

Comment on "Reintroduction of the archaic variant of in cortical organoids alters neurodevelopment".

Trujillo . (Research Articles, 12 February 2021, eaax2537) conclude that the reintroduction of an ancestral amino acid substitution in the protein NOVA1 drastically alters the development of brain organoids. We show that cell lines used by the authors carry heterozygous deletions of the target DNA sequence, providing another plausible explanation for the effects observed.

A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2.

SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may introduce the infection. Here we present a cheap and fast screening method based on direct RT-qPCR to detect SARS-CoV-2 in single or pooled gargle lavages ("mouthwashes"). This method detects individuals with large viral loads (Ct≤29) and we use it to test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility.

Simultaneous precise editing of multiple genes in human cells.

When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).