Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines. Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The mechanism of CRISPR/Cas-9 genome editing contains three steps, recognition, cleavage, and repair. The designed sgRNA recognizes the target sequence in the gene of interest through a complementary base pair. While the Cas-9 nuclease makes double-stranded breaks at a site 3 base pair upstream to protospacer adjacent motif, then the double-stranded break is repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. The CRISPR/Cas-9 genome-editing tool has a wide number of applications in many areas including medicine, agriculture, and biotechnology. In agriculture, it could help in the design of new grains to improve their nutritional value. In medicine, it is being investigated for cancers, HIV, and gene therapy such as sickle cell disease, cystic fibrosis, and Duchenne muscular dystrophy. The technology is also being utilized in the regulation of specific genes through the advanced modification of Cas-9 protein. However, immunogenicity, effective delivery systems, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications. Although CRISPR/Cas-9 becomes a new era in molecular biology and has countless roles ranging from basic molecular researches to clinical applications, there are still challenges to rub in the practical applications and various improvements are needed to overcome obstacles.

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Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing

Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1's PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.

Mb- and FnCpf1 nucleases are active in mammalian cells

Engineering a precise adenine base editor with minimal bystander editing

Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3.

Adenine base editors (ABE) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion However, the low activity of ABE at certain sites remains a major bottleneck that precludes efficacious applications Here, to address it, we develop a directional screening system in human cells to evolve the deaminase component of the ABE, and identify three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K) With further engineering, we create a consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibit superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax We also find that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve user-specified traits in base editors that extend beyond augmented editing activity

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Human cell based directed evolution of adenine base editors with improved efficiency.

Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.

Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity, which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editing activity and specificity of base editors (BEs) in human cells. Specifically, multiple cloning sites (MCS) were inserted into the human genome via lentivirus, and base editing targeting the MCS was performed with BEs. The base editing activities were assessed by specific restriction enzymes. The whole process only includes nucleotide-based targeting the MCS, editing, PCR, and digestion, thus, we named it NOTEPAD. This straightforward approach could be easily accessed by molecular biology laboratories. With this method, we could easily determine the BEs editing efficiency and pattern. The results revealed that BEs triggered more off-target effects in the genome than on plasmids including genomic indels (insertions and deletions). We found that ABEs (adenine base editors) had better fidelity than CBEs (cytosine base editors). Our system could be harnessed as a base editing assessment platform, which would pave the way for the development of next-generation BEs.

Development of a Simple and Quick Method to Assess Base Editing in Human Cells.

A precise and efficient adenine base editor.

Simple Summary JASMONATE ZIM-DOMAIN (JAZ) proteins are key components of the jasmonate (JA) signaling pathway in response to biotic and abiotic stresses in plants. Information about the JAZ gene family in pineapple (AcJAZ) is limited. In this study, 14 AcJAZ genes were identified in the pineapple genome. A complete overview of AcJAZ genes is presented, including the chromosome locations, phylogenetic relationships, gene structures, conserved motifs and cis-regulatory elements, and expression patterns at different developmental stages and under various stress conditions, and their possible involvement in diverse functions is suggested. Furthermore, the BiFC analysis revealed direct binary interactions between AcJAZs and crucial JA-signaling regulators in vivo. These results suggest that AcJAZs and other vital players function in the JA-signaling pathway in response to abiotic stresses in pineapple. Abstract JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of the jasmonate (JA)-signaling pathway and play pivotal roles in plant resistance to biotic and abiotic stresses. Genome-wide identification of JAZ genes has been performed in many plant species. However, systematic information about pineapple (Ananas comosus L. Merr.) JAZ genes (AcJAZs) is still not available. In this study, we identified 14 AcJAZ genes and classified them into five groups along with the Arabidopsis and rice orthologs. The AcJAZ genes have 3–10 exons, and the putative AcJAZ proteins have between two and eight conserved regions, including the TIFY motif and Jas domain. The cis-acting element analysis revealed that the putative promoter regions of AcJAZs contain between three and eight abiotic stress-responsive cis-acting elements. The gene-expression analysis suggested that AcJAZs were expressed differentially during plant development and subjected to regulation by the cold, heat, salt, and osmotic stresses as well as by phytohormones. Moreover, the BiFC analysis of protein interactions among the central JA-signaling regulators showed that AcJAZ4, AcMYC2, AcNINJA, and AcJAM1 could interact with AcJAZ5 and AcJAZ13 in vivo, indicating a canonical JA-signaling pathway in pineapple. These results increase our understanding of the functions of AcJAZs and the responses of the core players in the JA-signaling pathway to abiotic stresses.

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Investigation of the JASMONATE ZIM-DOMAIN Gene Family Reveals the Canonical JA-Signaling Pathway in Pineapple

The authors would like to make the following correction to the published paper [...].

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Correction: Ye et al. Investigation of the JASMONATE ZIM-DOMAIN Gene Family Reveals the Canonical JA-Signaling Pathway in Pineapple. Biology 2022, 11, 445

Xiaoxue He

Papers

Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.

Development of a Simple and Quick Method to Assess Base Editing in Human Cells.

A precise and efficient adenine base editor.

Investigation of the JASMONATE ZIM-DOMAIN Gene Family Reveals the Canonical JA-Signaling Pathway in Pineapple

Correction: Ye et al. Investigation of the JASMONATE ZIM-DOMAIN Gene Family Reveals the Canonical JA-Signaling Pathway in Pineapple. Biology 2022, 11, 445