Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Abstract

A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks (DSBs) which are then repaired by homology-directed repair (HDR) using synthetic or cloned DNA donor molecules carrying desired mutations. However, another pathway, the non-homologous end joining (NHEJ) pathway competes with HDR for repairing DNA breaks in cells. To increase the frequency of precise genome editing in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) we have tested the capacity of a number of small molecules to enhance HDR or inhibit NHEJ. We identify molecules that increase the frequency of precise genome editing including some that have additive effects when applied together. Using a mixture of such molecules, the 'CRISPY' mix, we achieve 2.8-to 6.7-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes, to our knowledge the highest editing efficiency in hiPSCs described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.9-to 4.0-fold, allowing almost 20% of chromosomes to be edited. Main textThe bacterial nuclease CRISPR/Cas9 is now frequently used to accurately cut chromosomal DNA sequences in eukaryotic cells. The resulting DNA breaks are repaired by two competing pathways: NHEJ and HDR (Fig. 1). In NHEJ, the first proteins to bind the cut DNA ends are Ku70/Ku80, followed by DNA protein kinase catalytic subunit (DNA-PKcs) Since NHEJ of Cas9-induced DSBs is error prone and frequently introduces short insertions and deletions (indels) at the cut site, it is useful for knocking out a targeted gene. In contrast, HDR allows precise repair of a DSB by using a homologous donor DNA. If the donor DNA provided in the experiment carries mutations, these will be introduced into the genome (precise genome editing). Repair with homologous ssDNA or dsDNA has been suggested to engage different pathways 5 . We will refer to Targeted Nucleotide Substitutions using ssDNA donors as 'TNS' and targeted insertion of cassettes . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/136374 doi: bioRxiv preprint first posted online May. 10, 2017; 3 using dsDNA donors as knock-ins, respectively. In order to introduce a DSB Cas9 requires the nucleotide sequence NGG (a "PAM" site) in the target DNA. Targeting of Cas9 is further determined by a guide RNA (gRNA) complementary to 20 nucleotides adjacent to the PAM site. However, the Cas9 may also cut the genome at sites that carry sequence similarity to the gRNA 6 . One strategy to reduce such off-target cuts is to use a mutated Cas9 that introduces single-stranded nicks instead of DSBs (Cas9n) 7 . Using two gRNAs to introduce two nicks on opposite DNA strands in close proximity to each other will result in a staggered D...

“Global Transcriptional Response to CRISPR/CAS9-AAV6 Based Genome Editing” Matches Transcriptional Response to Specific Small Molecule Perturbations

“…Separately, we found that the histone lysine methyltransferase inhibitor UNC-0321 shows negative connectivity to Cas9 mRNA or AAV and the DNA methyltransferase inhibitor zebularine has negative connectivity to AAV (Supplementary Table S2). Consistent with these observations, trichostatin was recently shown to enhance CRISPR/Cas9 enabled targeted nucleotide substitutions [13]. We provide more information on the top 20 molecules with negative connective score to each of the components in Supplementary Table S2.…”

“…Our results suggest that small molecules with shared transcriptional responses to the various gene editing components may influence distinct aspects of these processes. For example, some of the small molecules identified by our approach have been shown to influence CRISPR/Cas9 editing efficiency through their effects on distinct DNA repair processes: while trichostatin – an HDACi – enhances targeted nucleotide substitutions in CRISPR/Cas9 edited cells [13], L755507 enhances HDR [12]. Thus, small molecules could offer a means to fine-tune specific cellular processes associated with gene editing technologies.…”

Enhancing CRISPR deletion via pharmacological delay of DNA-PK

“…We hypothesised that successful CRISPR-del requires paired DSBs to co-occur before NHEJ has time to act, and thus may be enhanced by pharmacological inhibition of DNA-PK. This is initially counter-intuitive, as DNA-PK is a necessary step in the NHEJ pathway upon which CRISPR-del relies, and its inhibition is widely used to promote HDR 37,40,44,45 . However, rather than permanently blocking NHEJ, our protocol slows the kinetics of NHEJ for a defined period while DSBs are taking place.…”

“…Here, editing events are rare, and HR is the rate-limitingstep 38,39 . The two principal strategies to boost efficiency are: (1) direct stimulation of homology directed repair (HDR) 37,40-42 ; (2) suppression of the competing NHEJ pathway at early stages through inhibition of Ku70/80 complex 37,40,43 or DNA-dependent protein kinase (DNA-PK) 37,40,44,45 , or at late phases, via Ligase IV (LigIV) inhibition 37,40,46,47 . To date, however, there are no reported methods for pharmacological enhancement of CRISPR-del.…”

Improvement of the knock-in effciency in the genome of human induced pluripotent stem cells using the CRISPR/Cas9 system

“…Еще одна нуклеаза, делающая разрывы с липки ми концами, -Cpf1 (Zetsche et al, 2015). Сочетание ис поль зования SpCas9n или AsCpf1 с обработкой клеток несколькими органическими соединениями позволяет увеличить эффективность внесения целевой модификации в геном чИПСК в 3-7.2 раза, в зависимости от редакти руемого локуса и используемого сочетания (Ma et al, 2018;Riesenberg, Maricic, 2018).…”