Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

sgRNA design for DLT gene editing using CRISPR-Cas9 and in-silico mutation prediction in Rice cv. Hawara Bunar

The DWARF AND LOW TILLERRING (DLT) gene is a transcription factor for a gene involved in Brassinosteroid (BR) biosynthesis. Manipulating BR biosynthesis will affect the height and tiller number of rice. CRISPR-Cas9 is an accurate tool to edit a gene sequence. The accuracy of site editing of the CRISPR-Cas9-mediated target gene editing is determined by the 20 nucleotide sequences in the sgRNA and the binding site known as the Protospacer Adjacent Motif (PAM). The study aimed to design sgRNA and predict the DLT gene mutation sites in rice cv. Hawara Bunar. The exon 1 of the DLT gene was amplified using a primer pair designed from the reference gene. The PCR product was then sequenced, and the sequence was used to design sgRNA. The study has designed sgRNA located on the targeted sequence that corresponds to the Gras family protein domain of the exon1 DLT gene. The mutation sites were predicted to be at the domain site through the alignment of the nucleotide and amino acid sequences of the DLT gene and the reference gene. It is predicted that mutations in the target site that corresponds to the protein domain will change the protein structure and its function.

Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

Prime editing (PE) enables efficiently targeted introduction of multiple types of small-sized genetic change without requiring double-strand breaks or donor templates. Here we designed a simple strategy to introduce random DNA sequences into targeted genomic loci by prime editing, which we named random prime editing (Random-PE). In our strategy, the prime editing guide RNA (pegRNA) was engineered to harbor random sequences between the primer binding sequence (PBS) and homologous arm (HA) of the reverse transcriptase templates. With these pegRNAs, we achieved efficient targeted insertion or substitution of random sequences with different lengths, ranging from 5 to 10, in mammalian cells. Importantly, the diversity of inserted sequences is well preserved. By fine-tuning the design of random sequences, we were able to make simultaneously insertions or substitutions of random sequences in multiple sites, allowing in situ evolution of multiple positions in a given protein. Therefore, these results provide a framework for targeted integration of random sequences into genomes, which can be redirected for manifold applications, such as in situ protospacer adjacent motif (PAM) library construction, enhancer screening, and DNA barcoding.

RNA-guided AsCas12a- and SpCas9-catalyzed knockout and homology directed repair of the omega-1 locus of the human blood fluke, Schistosoma mansoni

We compared the efficiency and precision of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with SpCas9 of Streptococcus pyogenes aiming to advance functional genomics tools for Schistosoma mansoni. Programmed double stranded cleavage catalyzed by AsCas12a results in a staggered strand break whereas SpCas9 produces a blunt ended chromosomal break. The TTTV, the optimal protospacer adjacent motif for AsCas12a is expected frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions (gRNA:SpCas9:DNA donor ratio and electroporation condition) to edit the ω1 gene. SpCas9 delivered higher efficiency to mutate ω1 target compared to AsCas12a for non-homology end joining (NHEJ)-catalyzed repair (14.04% vs. 10.88%, n = 7 replicates). Most mutations were deletions; SpCas9 induced -3 nt size deletions whereas AsCas12a induced deletions ranging in size from -25 to -2 nt. Although these were less absolute percentage AsCas12a than SpCas9 programmed mutations, the phenotypic outcomes on levels of ω1 transcripts and expressed omega-1 protein were similar. Gene editing efficiency of SpCas9 and AsCas12a markedly increased in the presence of short single stranded donor templates bearing symmetrical homolog arms of 50 nt in length. With AsCas12a, both non-CRISPR target (NT) and target (T) strands of the ω1 gene were tested as homology direct repair (HDR) donor templates. There were 15.67%, 28.71% and 21.43% of NHEJ from 7 pooled genomic DNA from KI_SpCas9, KI_AsCas12a-NT-ssODN and KI_AsCas12a-T-ssODN experiments, respectively. Programmed SpCas9 cleavage led to higher levels than AsCas12a of precise HDR mediated; 17.07%, KI_SpCas9 vs. 14.58%, KI_AsCas12a-NT-ssODN and 12.35%, KI_AsCas12a-T-ssODN (P < 0.0.5), although no significant differences in reduction in ω1 transcripts or of protein levels were apparent. These findings revealed that both AsCas12a and SpCas9 can provide programmed knockout and transgene insertion into genes expressed in the schistosome egg.

Engineered PAM-flexible FnCas9 variants for robust and specific genome editing and diagnostics

The clinical success of CRISPR therapies is dependent on the safety and efficacy ofCas proteins. The Cas9 from Francisella novicida (FnCas9) has negligible affinity formismatched substrates enabling it to discriminate off-targets in DNA with very highprecision even at the level of binding. However, its cellular targeting efficiency is low,limiting its use in therapeutic applications. Here, we rationally engineer the protein todevelop engineered(enFnCas9) variants with enhanced activity and expand its cellularediting activity to genomic loci previously inaccessible. Notably, some of the variantsrelease the protospacer adjacent motif (PAM) constraint from NGG to NGR/NRGmaking them rank just below SpCas9-RY and SpCas9-NG in their accessibility acrosshuman genomic sites. The enFnCas9 proteins, similar to Cas12a and Cas12f, harborhigh intrinsic specificity and can diagnose single nucleotide variants accurately.Importantly, they provide superior outcomes in terms of editing efficiency, knock-inrates, and off-target specificity over other engineered high-fidelity versions of SpCas9(SpCas9-HF1 and eSpCas9). Broad targeting range coupled with remarkablespecificity of DNA interrogation underscores the utility of these variants for safe andefficient therapeutic gene correction across multiple cell lines and target loci.

PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems

Background The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. Results By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders. Conclusions This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.

CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences

RNA-guided genomic transcriptional regulation tools, namely clustered regularly interspaced short palindromic repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for gene functional studies. Deriving from the CRISPR/Cas9 system, both systems consist of a catalytically dead Cas9 (dCas9), a transcriptional effector and a single guide RNA (sgRNA). This type of dCas9 is incapable to cleave DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, protospacer adjacent motif (PAM) sequence requirements, efficient delivery methods and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.

Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

CRISPR-Cas9 bends and twists DNA to read its sequence

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

Mutation-specific reporter for optimization and enrichment of prime editing

Prime editing is a versatile genome-editing technique. However, some genomic sites remain difficult to edit and optimal design of prime-editing tools is elusive. We present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranked the efficiency of prime edit guide RNAs (pegRNAs) and any prime editor protein, including novel variants with flexible protospacer adjacent motif (PAM) recognition. We applied fluoPEER to instruct correction of pathogenic variants in patient cells and found that plasmid-editing enriched for editing at the genomic target site. Transcriptomic analysis of reporter-edited cells revealed that successful prime editing was associated with expression of DNA repair genes. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.