Our technology in more detail

PRCISR™ CRISPR libraries are produced using Vivlion’s proprietary Covalently-Closed-Circular-synthesized (3Cs) technology, enabling PCR-free and cloning-free generation of high-quality CRISPR/Cas gRNA libraries.

gRNA-encoding oligonucleotides are annealed to circular, deoxyuridine (dU)-containing single-stranded PRCISR™ CRISPR template plasmids, followed by their polymerase-mediated extension to form heteroduplexed double-stranded PRCISR™ CRISPR plasmids. Library diversity is defined solely by the number of gRNA-encoding oligonucleotides. PRCISR™ CRISPR libraries are amplified in bacteria, removing the template plasmids and yielding the final PRCISR™ CRISPR gRNA library.

The PRCISR™ CRISPR process that leads to uniform libraries of low skew is summarized in the following graph. This is contrasted with conventional library preparation that has the challenges of PCR amplification bias and template switching issues, in addition to restriction and ligation artefacts, leading to ununiform libraries with high distribution skews.

PRCISR™ CRISPR plasmids are offered in 3 possible gRNA arrangements: single, fixed-pair and dual-multiplex. All plasmids are available as all-in-one systems containing the nuclease and, in addition, are customizable according to choice of promoter, tracrRNA, antibiotic selection marker, fluorescence, barcode and scRNA-seq capture sequence.

Uniformity & skew

The generation of all CRISPR gRNA libraries starts with the choice of oligonucleotide pool that encodes the respective gRNA sequences.

Conventional cloning requires the PCR amplification of the oligonucleotide pool. During each cycle of the PCR amplification process, the polymerase amplifies certain gRNA sequences more than others which leads to under-represented and over-represented sequences (“sequence specific bias”) [REF]. In addition, the polymerase has a specific error rate which causes mutations in the amplified PCR product. Both of these issues are ultimately reflected in the final library’s gRNA sequences. After PCR amplification, the oligo pool is then incorporated into the plasmid using restriction and ligation cloning. This step is inefficient due to inefficient vector ligation and vector religation issues (“cloning artifacts”). Finally, the cloned library is amplified in bacteria to obtain the desired amounts of plasmid DNA – however, the uniformity of the library is impacted by sequence specific bias and cloning artifacts which lead to high skew.

In contrast, Vivlion’s 3Cs technology avoids these issues. 3Cs directly utilizes the oligo pool “as-is” (avoiding sequence specific PCR bias). In addition, the technology does not use restriction and ligation based cloning (avoiding cloning artifacts). The library is amplified in bacteria using highly efficient protocols to obtain the final highly uniform PRCISR™ CRISPR library with low skew (reflecting the distribution of the original oligo pool). 

How often do we see gRNAs occurring in the final library? A histogram shows how often gRNA sequences occur in the final library. The x-axis shows gRNA abundance and the y-axis shows the number of gRNAs with a specific abundance. Ideally, all gRNA sequences are equally abundant.

Editing efficiency

The gRNAs selected for many conventional CRISPR libraries are typically based on computational predictions of their activity. The highest scoring gRNAs are assembled into a library that delivers a single gRNA to a cell, resulting in a single gene editing event. For knockout-experiments, the introduction of a single insertion or deletion (InDel) leads to gene editing efficiencies of approximately 66%.

However, the gRNAs selected for Vivlion’s fixed-pair libraries are based on published data of hundreds of CRISPR screens. From this data, gRNAs are selected and assembled into a library of pre-defined pairs, delivering two highly functional gRNAs to each cell, each targeting a gene of interest. The gRNA pairs are designed to excise and remove a part of the target gene, thereby improving gene editing efficiencies to more than 90%.

Resources

Publications

Presentations at conferences and online series