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Methods to drastically increase precise genome editing efficiency with CRISPR enzymes in stem cells and various mammalian cells

Dr Tomislav Maricic, Max-Planck-Institute for Evolutionary Anthropology;  Stephan Riesenberg, Max-Planck-Institute for Evolutionary Anthropology

Max Planck Innovation


Challenge

The bacterial nuclease CRISPR/Cas9 allows to accurately cut chromosomal DNA sequences in eukaryotic cells. The induced DNA double-strand breaks (DSB) are repaired by two competing pathways: Non-homologous end joining (NHEJ) and homology-directed repair (HDR). However, NHEJ is an error-prone DNA repair pathway and is therefore used for gene inactivation rather than for precise genome editing (PGE). Moreover, by nature the rate of NHEJ is more efficient and thus is limiting the rate of PGE. Several attempts have been done to increase genome-editing efficiency either by enhancement of HDR or by inhibiting NHEJ, for example, by synchronization of cells to favor HDR repair over NHEJ, or by blocking proteins involved in NHEJ with siRNA or with small molecule inhibitors.


Technology

Scientists at the Max-Planck Institute for Evolutionary Anthropology in Leipzig have developed several methods to enhancing PGE efficiency in human induced pluripotent stem cells (hiPSC), embryonic stem cells (hESC) and blood cells (CD34+, CD4+):
A drastic increase of up to 19-fold or up to 90% precisely edited chromosomes with no additional indels has been achieved by applying a mutated repair protein and a small molecule cocktail, named CRISPY Mix, by directing DNA repair to HDR. To our knowledge, this is the highest efficiency ever reported in mammalian cells.

On the mutated line we performed multiplexed precise genome editing – a first time achievement in CRISPR editing of animal cells (33% of the clones we isolated were repaired with HDR homozygously on three targeted genes; we currently test if even more than three genes can be multiplexed). Using single stranded donor DNA, precise point mutations and gene fragments can be incorporated with unprecedented efficiencies. Furthermore, using the same strategy one could introduce targeted stop codons to inactivate the gene of interest and achieve better efficiencies than when a classical NHEJ knockout is used (when at best 66% of chromosomes carry frameshift mutations).


Commercial Opportunity

Kit, reagents and/or cell lines


Development Status

Product optimization required


Patent Situation

Two European priority patent applications have been filed in 2017.


Further Reading

Riesenberg and Maricic, in review


 

Methods to drastically increase precise genome editing efficiency with CRISPR enzymes in stem cells and various mammalian cells
Methods to drastically increase precise genome editing efficiency with CRISPR enzymes in stem cells and various mammalian cells

Figure 1. Inventions to radically increasing precise genome editing. The repair protein mutant increases HDR efficiency and often almost abolishes NHEJ frequency with Casn double nicking (A) and electroporation of Cas9 or Cpf1 (B) in induced pluripotent stem cells (PSCs). The stem cell CRISPY mix further increases HDR efficiency leading to 90% of HDR for some genes (C) in induced PSCs. The blood cell CRISPY mix increases HDR in CD34+ progenitor cells and CD4+ T cells (D). HDR, HDR + NHEJ, and NHEJ are indicated in green, light green and blue, respectively. Error bars show standard deviation of at least two replicate experiments.