Genetic Engineering Publications - GEG Tech top picks

Researchers have developed a new genetic system to test and analyze the mechanisms underlying the results of CRISPR-based DNA repair. As described in Nature Communications, they have developed a sequence analyzer to help track on- and off-target mutational changes and how they are inherited from one generation to the next. The tool, called Integrated Classifier Pipeline (ICP), can reveal specific categories of mutations resulting from CRISPR editing. Developed in flies and mosquitoes, ICP provides a "fingerprint" of how genetic material is inherited, enabling scientists to track the source of mutational changes and the associated risks emerging from potentially problematic modifications. PKI can help unravel the complex biological issues that arise when determining the mechanisms behind CRISPR. Although developed in insects, ICP has great potential for human applications.

An evolution of the CRISPR-Cas9 gene editing technology, master editing has enormous potential to treat genetic diseases in humans. However, to date, the factors determining successful editing are not well understood. A new tool to predict the chances of successfully inserting a gene-edited DNA sequence into the genome of a cell has been developed by researchers. The study, published in Nature Biotechnology, evaluated thousands of different DNA sequences inserted into the genome using master editors. This data was then used to train a machine learning algorithm to help researchers design the best solution for a given genetic defect, promising to speed up efforts to introduce master editing into the clinic. Sequence length proved to be a key factor, as was the type of DNA repair mechanism involved. The next steps for the team will be to create models for all known human genetic diseases to better understand if and how they can be corrected using master editing.

The race to the clinic is reviving for a ready-made alternative to autologous CAR-T cell therapy, even as concerns about chromosomal abnormalities persist. The Advanced Regenerative Medicine Therapy designation, which makes the therapy eligible for accelerated approval, will also help remove a veil that has hung over standard CAR-T cell therapies since October, when the FDA put all trials of competitor Allogene Therapeutics on hold following the detection of a chromosomal abnormality in a patient who received ALLO-501A in a Phase 2 trial. The FDA's green light for CRISPR Therapeutics dispels broader concerns that the agency views this type of genotoxic safety event as an intractable problem for the entire class of allogeneic CAR-T therapies. Today, many companies are eliminating loci associated with the MHC-I to avoid host T cell recognition of transplanted CAR-T cells. Companies also equip their T cells with a variety of safety switches and performance enhancers.

However, as the complexity of the assembly increases, the risk of off-target effects also increases. This may be important from a safety perspective, given that most cancers lack unique antigens. Achieving rapid remission and re-dosing if necessary, can minimize the toxic effects that CAR-T cells can have on healthy tissues expressing the targeted antigen.

About half of all bacteria studied and 90% of archaea use CRISPR-Cas systems to defend themselves against phages. In addition, prokaryotes have developed multiple mechanisms to control the expression of the CRISPR-Cas gene so that the defense can be stopped or activated as needed. However, phages retaliate and attempt to counter the defense via a diverse set of anti-CRISPR proteins (Acrs) that inhibit CRISPR-Cas activity and thus help the phage to successfully attack bacteria. Acrs have been experimentally exploited to reduce off-target edition by reducing the time available for Cas nuclease activity and increase tissue-specific edition by controlling their presence in various tissues. Understanding mechanically how CRISPR is regulated could lead to the improvement of CRISPR as a genome editing tool. Haridha Shivram, post-doctoral fellow at the University of California at Berkeley, has the research objective to discover new regulators of CRISPR-Case systems. The most fascinating aspect for him is how the arms race between the host and its invasive mobile genetic elements can lead to unique molecular innovations that can both help boost immunity but also reveal their hidden vulnerabilities. These evolving strategies can help make the CRISPR toolbox even better.

Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find off-targets generally do not work in vivo or detect single-nucleotide changes. Three papers in this issue report new methods for monitoring gene-editing tools in vivo (see the Perspective by Kempton and Qi). Wienert et al. followed the recruitment of a DNA repair protein to DNA breaks induced by CRISPR-Cas9, enabling unbiased detection of off-target editing in cellular and animal models. Zuo et al. identified off-targets without the interference of natural genetic heterogeneity by injecting base editors into one blastomere of a two-cell mouse embryo and leaving the other genetically identical blastomere unedited. Jin et al. performed whole-genome sequencing on individual, genome-edited rice plants to identify unintended mutations. Cytosine, but not adenine, base editors induced numerous single-nucleotide variants in both mouse and rice.

UC Berkeley researchers have made CRISPR/cas9 system even more versatile by giving it an “on” switch, allowing users to keep the Cas9 gene editor turned off in all cells except its designated target.

Here, the scientists describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, they provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA.

The scientists recently reported Digenome-seq (digested genome sequencing), a method for in vitro identification of potential off-target sites, and They evaluated the specificity of CRISPR–Cas9 and CRISPR–Cpf1 endonuclease by whole-genome sequencing. Digenome-seq pinpoints the exact location of double-strand break (DSB) sites by recognizing specific patterns of aligned reads. However, the analysis pipeline presented in their previous report required extensive manual interaction and produced several large intermediate files, resulting in a long running time. Here, the scientists present a redesigned analysis tool for Digenome-seq data that runs on web browsers. The core algorithm of the tool is written in C++ and compiled to asm.js (http://asmjs.org/), a preoptimized subset of JavaScript. Users can instantly perform the complete analysis in an ordinary web browser (Supplementary Note 1) with fast execution speed without uploading any data to a server and without local tool installation. In their benchmark, the full analysis for 100 GB of BAM file took 3 h for whole analysis on Intel i5 3570k central processing unit in a single thread.

Here, the authors developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing.

CRISPR-Cas9 screens are powerful high-throughput tools but can be confounded by nuclease toxicity. Here, to test potential solutions to this issue, the scientists design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. Their results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

Here the scientists conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, they collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. They find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. They further demonstrate that the best predictions can significantly reduce the time spent on guide screening.

One of the drawbacks of genome editing is that there are growing concerns about mutations and off-target effects. Researchers then hypothesized that current editing protocols that use Cas9 cause excessive DNA cleavage, resulting in some of the mutations. To test this hypothesis, the researchers built a system called "AIMS" in mouse cells, which assessed Cas9 activity separately for each chromosome. Their results showed that the commonly used method was associated with very high editing activity. They determined that this high activity caused some of the undesirable side effects, so they looked for gRNA editing methods that could suppress it. They found that an additional cytosine extension at the 5' end of the gRNA was effective as a "safeguard" against overactivity and controlled DNA cleavage. As a result of this study, the first mathematical model of the correlation between various genome editing patterns and Cas9 activity was created that can maximize the desired editing efficiency by developing activity-regulating gRNAs with appropriate Cas9 activity.

A major drawback of CRISPR-Cas9-mediated genome editing is that not all guide RNAs (gRNAs) efficiently cleave target DNA. Although the heterogeneity of gRNA activity is well recognized, the exact way in which CRISPR-Cas9 activity is regulated is not fully understood. In a recent study, a team of researchers used an energy-based model to identify the mechanisms regulating CRISPR-Cas9 activity and specificity. The team investigated the binding free energy, Δ G H , of RNA-DNA interactions and the effects of energy changes during RNA-DNA hybridisation on the efficiency of 11,602 Cas9 gRNAs experimentally validated in other studies. The efficiency of the gRNAs was measured as indel frequency, which is the most accurate indicator of CRISPR-Cas9 activity. They found that Cas9 'slippage' or binding competition on adjacent overlapping protospacer motifs (PAMs) affected gRNA activity by regulating local RNA-DNA interactions. Furthermore, the binding free energy sweet spot quantitatively defines the optimal activity of CRISPR-Cas9 at target and off-target sites in that the gRNA does not bind too weakly or too strongly. In the future, the team will continue to improve their methods, which will include further optimization of the gRNA design for increased efficiency and specificity. 

A group of researchers set out to characterize the genotypic abnormalities induced by Cas9 in human cells. To do this, they studied the tRNA gene. These researchers deleted two tRNA genes from the genomes of hyperploid human hepatocellular carcinoma (HepG2) and haploid chronic myeloid leukemia (HAP1) cells using the CRISPR/Cas9 system with dual gRNAs. The underlying alterations that caused rearrangement of the CRISPR/Cas9-targeted region were assessed by a customized de novo sequence assembly approach. Although a genomic region of interest was cleaved by Cas9 in this study, the cleaved fragment was duplicated, inverted, and inserted locally into the genomes of both HepG2 and HAP1 cells. The study also demonstrated the successful integration of exogenous DNA fragments into HepG2 cells.

Furthermore, the aberrant target-derived DNA fragments were shown to be still functional, tagged with active histones, and bound by RNA polymerase III. This highlights the fact that CRISPR/Cas9-based genomic engineering can lead to adverse effects on the target, and that inversion and duplication events can occur at the same time. In conclusion, this study reveals the complex genomic alterations that accompany CRISPR/Cas9 deletions.

In this article, the scientists  present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. They pave the way for a single-molecule system, LEAPER, enabling precise, efficient RNA editing with broad applicability for therapy and basic research.

Researchers have engineered another CRISPR system, Cas12b, which offers improved capabilities and options when compared to CRISPR-Cas9 systems. The work was led by Feng Zhang of the Broad Institute of MIT and Harvard, the McGovern Institute, and MIT.

Cas9-induced double stranded breaks can cause large deletions near the target site and more complex genomic rearrangements in mouse and human stem cells.

“Unexpected mutations” simply represent SNPs and indels shared in common by these mice prior to nuclease treatment.

What is your opinion?

To the Editor: CRISPR–Cas9 editing shows promise for correcting
disease-causing mutations. For example, in a recent study we
used CRISPR-Cas9 for sight restoration in blind rd1 mice by correcting
a mutation in the Pde6b gene1. However, concerns persist
regarding secondary mutations in regions not targeted by the
single guide RNA (sgRNA)2. Algorithms generate likely off-target
sites for a given gRNA, but these algorithms may miss mutations.
Whole-genome sequencing (WGS) has been used to assess the
presence of small insertions and deletions (indels)3 but not to
probe for single-nucleotide variants (SNVs) in a whole organism.
We performed WGS on a CRISPR–Cas9-edited mouse to
identify all off-target mutations and found an unexpectedly high
number of SNVs compared with the widely accepted assumption
that CRISPR causes mostly indels at regions homologous to the
sgRNA.

The methods present in this article decouple aspects of kinetic and thermodynamic properties of the Cas9–DNA interaction and broaden the toolkit for investigating off-target binding behavior.

CIRCLE-seq is an in vitro assay for selectively sequencing off-target sites cleaved by Cas9-sgRNA in genomic DNA. It sensitively profiles genome-wide off-target cut sites and characterizes the contribution of SNPs to these cleavage events.

In this study, the scientists introduce microRNA sponge generator and tester (miRNAsong), a freely available web-based tool for generation and in silico testing of miRNA sponges. This tool generates miRNA sponge constructs for specific miRNAs and miRNA families/clusters and tests them for potential binding to miRNAs in selected organisms. Currently, miRNAsong allows for testing of sponge constructs in 219 species covering 35,828 miRNA sequences.

This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.