Improved sgRNA Design Delivers Better CRISPR Knockout Screen Results

Last year, Cellecta received a Phase 1 NIH SBIR grant to develop validated single guide RNA (sgRNA). The initial study was designed to investigate an approach to optimize CRISPR sgRNA to improve the efficiency of CRISPR knockout.

The 5'-end of the CRISPR sgRNA contains 20 nucleotides specific to the gene targeted for knockout. The rest of the sgRNA—the 3' end—interacts with the Cas9 nuclease to catalyze the DNA break and generate the knockout. There's been significant effort to optimize the design of the 5'-targeting sequences to make more effective guides. However, modifications to the constant 3'-end of sgRNA have been shown to enhance Cas9 binding and improvements in this region, if they result in more effective knockout, could be applied universally across all sgRNA regardless of the target.

At the start of the grant, we showed that we could improve the rate and efficiency of sgRNA knockout of a GFP target gene with a few modifications to the constant 3'-region. Similar results where also shown recently by Dang et al. We looked at three changes in particular: one swaps the places of an adenine (A) and thymine (T) to remove a transcription terminator site. Another alternation adds a 5 nucleotide extension ("HE") to the stem of a stem-loop structure which should make it more stable and accessible to the Cas9 protein. For the third, we combined the two HE and AT modifications (HEAT).

Over the past several months, we extended this work to investigate whether these sgRNA modifications would confer significant measurable improvement for genetic screening. This required running actual genetic screens on numerous targets with multiple guides so we built four pooled libraries: one each with the AT, HE, and HEAT changes used for the GFP knockout experiment above and one with the standard design ("wt") as described by Jinek, et al. We then ran "dropout viability" screens with these pooled CRISPR libraries to identify essential genes. In this sort of screen, cells harboring sgRNA that knock out genes necessary for cell viability either die or do not proliferate, and so, these guides become depleted after a period of time.

As you can see from the "waterfall" plots below, the positive control sgRNAs—the sgRNAs that target known essential genes—are more consistently and strongly depleted in the screens with the libraries that contained the modified sgRNA designs. In fact, Z‑score analysis of depletion levels of 8 sgRNA to 20 different essential genes showed consistent increase in the magnitude of the signal for the sgRNA containing the modifications, with the library that included both the AT inversion and HE insertion having the most substantial improvement in dropout. You can see more details about this study in the following Application Note.

The findings clearly show that it is possible to substantially improve the quality of sgRNA libraries with just a few changes in the constant 3'-sequence of sgRNAs. Libraries with this modified sgRNA structure knock out target genes more quickly and effectively. Cellecta routinely uses the HEAT-modified sgRNA design in its constructs and libraries, including our CRISPR Human Genome Knockout Library.

CRISPR sgRNA tracrRNA design optimization screens

The graphs show depletion levels of sgRNA from four libraries, each targeting the same set of genes with a different variation of the sgRNA 3'-sequence: (A) the "wild type" (standard) sequence, (B) an AT inversion that eliminates a transcription termination site, (C) an insertion (HE) that elongates and stabilizes a stem-loop structure, and (D) both the HE and AT (HEAT) modifications. The positive controls that target essential genes (lines in red) are more strongly depleted in the libraries with the modified sgRNA structures. Further Z-score analysis of depletion levels of all 8 sgRNA targeting each of 20 different essential positive control genes show that depletion levels of the HEAT-modified sgRNA were the most consistent and robust of the four variations.

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