Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.
Highlights
The emergence of Clustered Regularly Interspaced Short Palindromic Repeats and their associated nucleolytic enzymes (CRISPR/Cas) have revolutionized our capacity to execute genomic engineering in eukaryotic and prokaryotic cells [1,2,3,4,5]
As the CRISPR/Cas gene editing technology continues to dominate the genetic engineering of mammalian cells, efforts are underway to elucidate the reaction parameters that surround its precision and efficiency
Previous studies have focused on the degree of homology between the CRISPR guide
Summary
The emergence of Clustered Regularly Interspaced Short Palindromic Repeats and their associated nucleolytic enzymes (CRISPR/Cas) have revolutionized our capacity to execute genomic engineering in eukaryotic and prokaryotic cells [1,2,3,4,5]. CRISPR/Cas can be used to disable functioning genes through the activation of a cellular process known as Non-Homologous End Joining (NHEJ) [2,6]. This DNA damage repair pathway is activated when CRISPR/Cas makes specific double-strand breaks (DSBs) in DNA. As enzymatic processing of the broken DNA ends occurs, single or multiple nucleotides on either or both strands can be lost [6,7] These changes can result in the alteration of the reading frame and lead to the generation of so-called genetic knockouts through targeted gene editing
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