Molecular therapy. Nucleic acidsApr 25, 2020

CRISPR/Cas tolerance for mismatched guide RNA sequences during precise DNA repair in the lab

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 3, 2026

Abstract

CRISPR-directed gene editing shows a surprisingly high tolerance for mismatches between guide RNA and target DNA.

Gene editing relies on accurate pairing between guide RNA and target DNA for successful outcomes.
Variation in guide RNA sequences may impact the effectiveness of CRISPR technology across different genetic backgrounds.
Nonhomologous end joining and homology-directed repair can produce a wide range of gene editing results.
Heterologous binding sites allow for both precise and error-prone gene editing outcomes.
Mispairing between guide RNA and target DNA can lead to unintended genomic editing activities.

Simplified

CRISPR and associated Cas nucleases are genetic engineering tools revolutionizing innovative approaches to cancer and inherited diseases. CRISPR-directed gene editing relies heavily on proper DNA sequence alignment between the guide RNA (gRNA)/CRISPR complex and its genomic target. Accurate hybridization of complementary DNA initiates gene editing in human cells, but inherent gRNA sequence variation that could influence the gene editing reaction has been clearly established among diverse genetic populations. As this technology advances toward clinical implementation, it will be essential to assess what degree of gRNA variation generates unwanted and erroneous CRISPR activity. With the use of a system in which a cell-free extract catalyzes nonhomologous end joining (NHEJ) and homology-directed repair (HDR), it is possible to observe a more representative population of all forms of gene editing outcomes. In this manuscript, we demonstrate CRISPR/Cas complexation at heterologous binding sites that facilitate precise and error-prone HDR. The tolerance of mispairing between the gRNA and target site of the DNA to enable HDR is surprisingly high and greatly influenced by polarity of the donor DNA strand in the reaction. These results suggest that some collateral genomic activity could occur at unintended sites in CRISPR-directed gene editing in human cells.

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CRISPR/Cas tolerance for mismatched guide RNA sequences during precise DNA repair in the lab

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