Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering

Abstract

Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5’ fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA. The tolerance of the gRNA and donor DNA to chemical modifications has the potential to enable new strategies for genome engineering.DOI: http://dx.doi.org/10.7554/eLife.25312.001

Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

“…37). Future work is required to study other types of chemical modifications or incorporation of DNA aptamers at the 5′ end of crRNA and sgRNA 38 . In addition, whether DNA Cpf1 crRNA can decrease off-target specificity has yet to be tested.…”

Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

“…By contrast, it has been very difficult to efficiently edit primary cells, which commonly undergo apoptosis, and/or preferentially employ NHEJ for damage repair, leading to a high ratio of indels to precise genome edits 1 . Several efforts have been made to improve genome editing of primary cells, including increasing the concentration of the repair DNA template, delivering NHEJ inhibitors, and optimizing transfection [1][2][3][4][5] . Although these means can improve precision editing up to threefold, a mechanistic explanation for its general inefficiency is lacking.…”

“Ancestralization” of human pluripotent stem cells by multiplexed precise genome editing

“…Several studies have therefore tried to inhibit NHEJ in order to increase the efficiencies of PGE, for example by blocking the synthesis of NHEJ proteins with siRNAs, by the expression of adenovirus proteins that degrade proteins necessary for NHEJ 8 , and by using small molecules that block NHEJ 9,10 . Others have tried to enhance HDR by cell cycle synchronization 11,12 and improved DNA donor design [13][14][15] . A third approach that does not rely on HDR to introduce nucleotide substitutions is to use proteins that chemically induce nucleotide transitions but this allows only four out of 12 possible substitutions to be introduced 16,17 .…”