Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair

Abstract

As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.

The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template

“…Out of 64 white colonies analyzed, 42 contained an exact insertion of the eight base fragment, while the remaining 22 colonies contained insertions ranging from 26 to 34 bases in length (see Figure S1 for additional sequences). This error-prone product likely involves NHEJ activity which, as previously demonstrated, occurs simultaneously in the in vitro reaction [ 15 ].…”

“…The in vitro, cell-free system used in these experiments has been previously established, through extensive control experimentation and replication to determine the optimal reaction parameters which prove the robustness and reproducibility of using this system [ 15 , 26 , 27 , 28 ]. Briefly ( Figure 2 A), the plasmid template is incubated with a CRISPR/Cas ribonucleoprotein (RNP) complex for 15 min to allow for complete cleavage of the LacZα gene containing plasmid molecules.…”

“…Some of these products display a precise insertion or precise correction of the targeted site whereas in other cases, multiple combinations of genetic changes at the DNA level are observed. Recently, Sansbury et al [ 15 ] began to define, catalog, and quantitate the diverse genetic population that resulted from CRISPR-directed gene editing using a mammalian cell-free extract that can support both NHEJ and HDR events simultaneously.…”

“…Several characteristics have been shown to play important roles in the design of donor DNA templates to increase the chances of successful genome editing. Strand bias is one characteristic in which one polarity of the donor DNA, designed to pair a relative strand of the DNA helix, outperforms the other [ 8 , 13 , 14 , 15 , 20 , 26 , 27 , 28 ]. In almost all cases, donor DNA templates that are complementary to the sense strand of the target site catalyze a higher degree of gene editing.…”

“…In addition, gene editing outcomes in this system are not influenced by transfection efficiency of intact cells or the degradation of the CRISPR/Cas complex as it transits to the nucleus [ 27 , 28 ]. This system has already partially elucidated the molecular activity of two CRISPR associated nucleases, Cas12a and Cas9 (CRISPR/Cas12a and CRISPR/Cas9) in both precise and error-prone HDR reaction pathways [ 15 , 27 ]. Our previous studies prompted us to consider the possibility that the length of ssDNA donor templates could also influence the diversity of genetic outcomes in both precise and error-prone gene editing reactions.…”