crispr-guide-rna-cloning-for-mammalian-systems

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

Harvard University

Harvard Medical School

Massachusetts General Hospital

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

Highly efficient Cas9-mediated transcriptional programming.

We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. 

Cas9 gRNA engineering for genome editing, activation and repression.

Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.

Comparison of Cas9 activators in multiple species.

It has been possible to create tools to predict single guide RNA (sgRNA) activity in the CRISPR/Cas9 system derived from Streptococcus pyogenes due to the large amount of data that has been generated in sgRNA library screens. However, with the discovery of additional CRISPR systems from different bacteria, which show potent activity in eukaryotic cells, the approach of generating large data sets for each of these systems to predict their activity is not tractable. Here, we present a new guide RNA tool that can predict sgRNA activity across multiple CRISPR systems. In addition to predicting activity for Cas9 from S. pyogenes and Streptococcus thermophilus CRISPR1, we experimentally demonstrate that our algorithm can predict activity for Cas9 from Staphylococcus aureus and S. thermophilus CRISPR3. We also have made available a new version of our software, sgRNA Scorer 2.0, which will allow users to identify sgRNA sites for any PAM sequence of interest.

sgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity.

The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.

Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.

Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR-Cas9-based 'gene drive array' platform to facilitate efficient genetic analysis in C. albicans. In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens.

A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans.

Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

Precise Cas9 targeting enables genomic mutation prevention.

Genetic manipulation of microorganisms has been crucial in understanding their biology, yet for many microbial species, robust tools for comprehensive genetic analysis were lacking until the advent of CRISPR-Cas-based gene editing techniques. In this Progress article, we discuss advances in CRISPR-based techniques for the genetic analysis of genetically intractable microorganisms, with an emphasis on mycobacteria, fungi and parasites. We discuss how CRISPR-based analyses in these organisms have enabled the discovery of novel gene functions, the investigation of genetic interaction networks and the identification of virulence factors.

CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms.

We describe a method that enables the multiplex screening of a pool of many different donor cell lines. Our method accurately predicts each donor proportion from the pool without requiring the use of unique DNA barcodes as markers of donor identity. Instead, we take advantage of common single nucleotide polymorphisms, whole-genome sequencing, and an algorithm to calculate the proportions from the sequencing data. By testing using simulated and real data, we showed that our method robustly predicts the individual proportions from a mixed-pool of numerous donors, thus enabling the multiplexed testing of diverse donor cells en masse.More information is available at https://pgpresearch.med.harvard.edu/poolseq/.

Enabling multiplexed testing of pooled donor cells through whole-genome sequencing.

Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.

An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance.

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

Article	Year
Highly efficient Cas9-mediated transcriptional programming. Nature methods\| PubMed ID: 25730490	2015
Cas9 gRNA engineering for genome editing, activation and repression. Nature methods\| PubMed ID: 26344044	2015
Comparison of Cas9 activators in multiple species. Nature methods\| PubMed ID: 27214048	2016
sgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity. ACS synthetic biology\| PubMed ID: 28146356	2017
Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nature microbiology\| PubMed ID: 28165460	2017
A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans. Nature microbiology\| PubMed ID: 29062088	2017
Precise Cas9 targeting enables genomic mutation prevention. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29555762	2018
CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms. Nature reviews. Microbiology\| PubMed ID: 29599458	2018
Enabling multiplexed testing of pooled donor cells through whole-genome sequencing. Genome medicine\| PubMed ID: 29673390	2018
An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance. Cell\| PubMed ID: 29677511	2018
High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast. Nature biotechnology\| PubMed ID: 29786095	2018
Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks. Molecular cell\| PubMed ID: 29979968	2018
An enhanced CRISPR repressor for targeted mammalian gene regulation. Nature methods\| PubMed ID: 30013045	2018

Alejandro Chavez

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