Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae.

We evaluated the Cre-lox and CRISPR-Cas9 systems as marker-recycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4-based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.

New opportunities for designing effective small interfering RNAs.

Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.

Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding-rewinding equilibrium. Increasing PAM-distal mismatches facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.

Assessment of External Guide Sequences' (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules.

RNase P is a ribozyme consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. It was initially identified as the enzyme that mediates cleavage of precursor tRNAs at the 5'-end termini to generate the mature tRNAs. An important characteristic of RNase P is that its specificity depends on the structure rather than the sequence of the RNA substrate. Any RNA species that interacts with an antisense molecule (called external guide sequence, EGS) and forms the appropriate structure can be cleaved by RNase P. This property is the basis for EGS technology, an antisense methodology for inhibiting gene expression by eliciting RNase P-mediated cleavage of a target mRNA molecule. EGS technology is being developed to design therapies against a large variety of diseases. An essential milestone in developing EGSs as therapies is the assessment of the efficiency of antisense molecules to induce cleavage of the target mRNA and evaluate their effect in vivo. Here, we describe simple protocols to test the ability of EGSs to induce cleavage of a target mRNA in vitro and to induce a phenotypic change in growing cells.

CRISPulator: a discrete simulation tool for pooled genetic screens.

BACKGROUND: The rapid adoption of CRISPR technology has enabled biomedical researchers to conduct CRISPR-based genetic screens in a pooled format. The quality of results from such screens is heavily dependent on the selection of optimal screen design parameters, which also affects cost and scalability. However, the cost and effort of implementing pooled screens prohibits experimental testing of a large number of parameters. RESULTS: We present CRISPulator, a Monte Carlo method-based computational tool that simulates the impact of screen parameters on the robustness of screen results, thereby enabling users to build intuition and insights that will inform their experimental strategy. CRISPulator enables the simulation of screens relying on either CRISPR interference (CRISPRi) or CRISPR nuclease (CRISPRn). Pooled screens based on cell growth/survival, as well as fluorescence-activated cell sorting according to fluorescent reporter phenotypes are supported. CRISPulator is freely available online ( http://crispulator.ucsf.edu ). CONCLUSIONS: CRISPulator facilitates the design of pooled genetic screens by enabling the exploration of a large space of experimental parameters in silico, rather than through costly experimental trial and error. We illustrate its power by deriving non-obvious rules for optimal screen design.

CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment.

The CRISPR/Cas9 complex, a bacterial immune response system, has been widely adopted for RNA-guided genome editing and transcription regulation in applications such as targeted genome modification and site-directed mutagenesis. However, the physical basis for its target specificity is not fully understood. In this study, based on a statistical mechanical analysis for the whole ensemble of sgRNA-target complex conformations, we identify a strong correlation between Cas9 cleavage efficiency and the stability of the DNA-RNA (R-loop) complex structures, with a Pearson correlation coefficient ranging from 0.775 to 0.886 for the tested systems. The finding leads to quantitative insights into important experimental results, such as the greater Cas9 tolerance to single-base mismatches in PAM-distal region than to PAM-proximal region and the high specificity and efficiency for shorter protospacers. Moreover, the results from the genome-wide off-target assessments, compared with other off-target scoring tools, indicate that the statistical mechanics-based approach provides more reliable off-target analyses and sgRNA design. To facilitate the genome engineering applications, a new web-based tool for genome-wide off-target assessment is established.

Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.