A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.

Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome, which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence. Thus, with the availability of the facile CRISPR/Cas9 gene editing tool it now seems easier to approach investigation of the functions of the glycome through genetic dissection of biosynthetic pathways, rather than by direct glycan analysis. However, obstacles still remain with design and validation of efficient gene targeting constructs, as well as with the interpretation of results from gene targeting and the translation of gene function to glycan structures. This is especially true for glycosylation steps covered by isoenzyme gene families. Here, we present a library of validated high-efficiency gRNA designs suitable for individual and combinatorial targeting of the human glycosyltransferase genome together with a global view of the predicted functions of human glycosyltransferases to facilitate and guide gene targeting strategies in studies of the human glycome.

Dynamics of Indel Profiles Induced by Various CRISPR/Cas9 Delivery Methods.

The introduction of CRISPR/Cas9 gene editing in mammalian cells is a scientific breakthrough, which has greatly affected basic research and gene therapy. The simplicity and general access to CRISPR/Cas9 reagents has in an unprecedented manner "democratized" gene targeting in biomedical research, enabling genetic engineering of any gene in any cell, tissue, organ, and organism. The ability for fast, precise, and efficient profiling of the double-stranded break induced insertions and deletions (indels), mediated by any of the available programmable nucleases, is paramount to any given gene targeting approach. In this study we review the most commonly used indel detection methods and using a robust, sensitive, and cost efficient Indel Detection by Amplicon Analysis method, we have investigated the impact of the most commonly used CRISPR/Cas9 delivery formats, including lentivirus transduction, plasmid lipofection, and ribo nuclear protein electroporation, on the dynamics of indel profile formation. We observe rapid indel formation using RNP electroporation, especially with synthetic stabilized gRNA, as well as long-term decline in overall indel frequency with lipofectamine-based, plasmid transfection methods. Most methods reach peak editing on day 2-3 postdelivery. Furthermore, we find relative increase in frequency of larger size indels (>6bp) under condition of persistent editing using stably integrated lentiviral gRNA and Cas9 vectors.