The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

http://knockout.cwru.edu/publications/hsucrisprreview.pdf

Development and applications of CRISPR-Cas9 for genome engineering.

Genome-wide, targeted loss-of-function pooled screens using the CRISPR (clustered regularly interspaced short palindrome repeats)–associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi) and have been used to reveal new mechanisms in diverse biological models1-4. Previously, we used a Genome-scale CRISPR Knock-Out (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, limiting the range of biological systems amenable to screening.

Here, we sought to improve both the lentiviral packaging and choice of guide sequences in our original GeCKO library1, where a pooled library of synthesized oligonucleotides was cloned into a lentiviral backbone containing both the Streptococcus pyogenes Cas9 nuclease and the single guide RNA (sgRNA) scaffold. To create a new vector capable of producing higher-titer virus (lentiCRISPRv2), we made several modifications, including removal of one of the nuclear localization signals (NLS), human codon-optimization of the remaining NLS and P2A bicistronic linker sequences, and repositioning of the U6-driven sgRNA cassette (Fig. 1a). These changes resulted in a ~10-fold increase in functional viral titer over lentiCRISPRv11 (Fig. 1b).



Figure 1

New lentiviral CRISPR designs produce viruses with higher functional titer.



To further increase viral titer, we also cloned a two-vector system, in which Cas9 (lentiCas9-Blast) and sgRNA (lentiGuide-Puro) are delivered using separate viral vectors with distinct antibiotic selection markers (Fig. 1a). LentiGuide-Puro has a ~100-fold increase in functional viral titer over the original lentiCRISPRv1 (Fig. 1b). Both single and dual-vector systems mediate efficient knock-out of a genomically-integrated copy of EGFP in human cells (Supplementary Fig. 1). Whereas the dual vector system enables generation of Cas9-expressing cell lines which can be subsequently used for screens using lentiGuide-Puro, the single vector lentiCRISPRv2 may be better suited for in vivo or primary cell screening applications.

In addition to the vector improvements, we designed and synthesized new human and mouse GeCKOv2 sgRNA libraries (Supplementary Methods) with several improvements (Table 1): First, for both human and mouse libraries, to target all genes with a uniform number of sgRNAs, we selected 6 sgRNAs per gene distributed over 3-4 constitutively expressed exons. Second, to further minimize off-target genome modification, we improved the calculation of off-target scores based on specificity analysis5. Third, to inactivate microRNAs (miRNAs) which play a key role in transcriptional regulation, we added sgRNAs to direct mutations to the pre-miRNA hairpin structure6. Finally, we targeted ~1000 additional genes not included in the original GeCKO library.



Table 1

Comparison of new GeCKO v2 human and mouse sgRNA libraries with existing CRISPR libraries.



Both libraries, mouse and human, are divided into 2 sub-libraries — containing 3 sgRNAs targeting each gene in the genome, as well as 1000 non-targeting control sgRNAs. Screens can be performed by combining both sub-libraries, yielding 6 sgRNAs per gene, for higher coverage. Alternatively, individual sub-libraries can be used in situations where cell numbers are limiting (eg. primary cells, in vivo screens). The human and mouse libraries have been cloned into lentiCRISPRv2 and into lentiGuide-Puro and deep sequenced to ensure uniform representation (Supplementary Fig. 2, 3). These new lentiCRISPR vectors and human and mouse libraries further improve the GeCKO reagents for diverse screening applications. Reagents are available to the academic community through Addgene and associated protocols, support forums, and computational tools are available via the Zhang lab website (www.genome-engineering.org).

Improved vectors and genome-wide libraries for CRISPR screening.

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

http://stacks.cdc.gov/view/cdc/29957/cdc_29957_DS1.pdf

CRISPR-Cas systems for editing, regulating and targeting genomes

CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.

Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12 , as well as novel hits NF2 , CUL3 , TADA2B , and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

/pdf/genome-scale-crispr-cas9-knockout-screening-in-human-cells-52hijutmcc.pdf

Xi-Shun Shi

Papers

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells