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MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

2021-03-22T00:00

CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.

Graphical Abstract

Target transcription activation with CjCas9-based gene activators. Three gene activators, SunTag-dCjCas9/VPR, VPR-dCjCas9, and VPR-CjCas9, are developed. Minimization and optimization of VPR-dCjCas9 leads to creation of MiniCAFE, which is able to activate gene expression and induce corresponding phenotypes in C. elegans, mice and human cells.

A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing

2021-01-27T00:00

Generation of conditional knockout (cKO) and various gene-modified cells is laborious and time-consuming. Here, we established an all-in-one cKO system, which enables highly efficient generation of cKO cells and simultaneous gene modifications, including epitope tagging and reporter gene knock-in. We applied this system to mouse embryonic stem cells (ESCs) and generated RNA helicase Ddx1 cKO ESCs. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. We further established TetFE ESCs, which carried a reverse tetracycline transactivator (rtTA) expression cassette and a tetracycline response element (TRE)-regulated FLPERT2 cassette in the Gt(ROSA26)Sor locus for instant and tightly regulated induction of gene KO. By utilizing TetFE Ddx1^F/F ESCs, we isolated highly pure Ddx1^F/F and Ddx1⁻^/⁻ ESCs and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress–p53 pathway. We also demonstrated cKO of various genes in ESCs and homologous recombination-non-proficient human HT1080 cells. The frequency of cKO clones was remarkably high for both cell types and reached up to 96% when EGFP-positive clones were analyzed. This all-in-one cKO system will be a powerful tool for rapid and precise analyses of gene functions.

A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs

2021-01-15T00:00

Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed ‘Self-targeting sgRNA Library Screen’ (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9.

Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects

2021-02-22T00:00

Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however, engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps. Finally, our REDIT method is applicable across cell types including human stem cells, and is generalizable to different Cas9 enzymes.

CRISPRidentify: identification of CRISPR arrays using machine learning approach

2020-12-08T00:00

CRISPR–Cas are adaptive immune systems that degrade foreign genetic elements in archaea and bacteria. In carrying out their immune functions, CRISPR–Cas systems heavily rely on RNA components. These CRISPR (cr) RNAs are repeat-spacer units that are produced by processing of pre-crRNA, the transcript of CRISPR arrays, and guide Cas protein(s) to the cognate invading nucleic acids, enabling their destruction. Several bioinformatics tools have been developed to detect CRISPR arrays based solely on DNA sequences, but all these tools employ the same strategy of looking for repetitive patterns, which might correspond to CRISPR array repeats. The identified patterns are evaluated using a fixed, built-in scoring function, and arrays exceeding a cut-off value are reported. Here, we instead introduce a data-driven approach that uses machine learning to detect and differentiate true CRISPR arrays from false ones based on several features. Our CRISPR detection tool, CRISPRidentify, performs three steps: detection, feature extraction and classification based on manually curated sets of positive and negative examples of CRISPR arrays. The identified CRISPR arrays are then reported to the user accompanied by detailed annotation. We demonstrate that our approach identifies not only previously detected CRISPR arrays, but also CRISPR array candidates not detected by other tools. Compared to other methods, our tool has a drastically reduced false positive rate. In contrast to the existing tools, our approach not only provides the user with the basic statistics on the identified CRISPR arrays but also produces a certainty score as a practical measure of the likelihood that a given genomic region is a CRISPR array.

Precise and broad scope genome editing based on high-specificity Cas9 nickases

2021-01-04T00:00

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9^D10A variants whose precision is superior to that of the commonly used Cas9^D10A nickase. Dual nicking RGNs based on a selected group of these Cas9^D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by ‘tiptoeing’ over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing

2021-01-04T00:00

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T₅₀ (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy

2020-12-03T00:00

The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.

A simplified strategy for titrating gene expression reveals new relationships between genotype, environment, and bacterial growth

2020-11-22T00:00

A lack of high-throughput techniques for making titrated, gene-specific changes in expression limits our understanding of the relationship between gene expression and cell phenotype. Here, we present a generalizable approach for quantifying growth rate as a function of titrated changes in gene expression level. The approach works by performing CRISPRi with a series of mutated single guide RNAs (sgRNAs) that modulate gene expression. To evaluate sgRNA mutation strategies, we constructed a library of 5927 sgRNAs targeting 88 genes in Escherichia coli MG1655 and measured the effects on growth rate. We found that a compounding mutational strategy, through which mutations are incrementally added to the sgRNA, presented a straightforward way to generate a monotonic and gradated relationship between mutation number and growth rate effect. We also implemented molecular barcoding to detect and correct for mutations that ‘escape’ the CRISPRi targeting machinery; this strategy unmasked deleterious growth rate effects obscured by the standard approach of ignoring escapers. Finally, we performed controlled environmental variations and observed that many gene-by-environment interactions go completely undetected at the limit of maximum knockdown, but instead manifest at intermediate expression perturbation strengths. Overall, our work provides an experimental platform for quantifying the phenotypic response to gene expression variation.

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics

2020-12-10T00:00

Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms – MENTHU, inDelphi, and Lindel – in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.