Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica.

CRISPR/Cas9, presently the most widely used genome editing technology, has provided great potential for functional studies and plant breeding. However, the strict requirement for a protospacer adjacent motif (PAM) has hindered the application of the CRISPR/Cas9 system because the number of targetable genomic sites is limited. Recently, the engineered variants Cas9-NG, SpG, and SpRY, which recognize non-canonical PAMs, have been successfully tested in plants (mainly in rice, a monocot). In this study, we evaluated the targeted mutagenesis capabilities of these Cas9 variants in two important Brassica vegetables, Chinese cabbage (Brassica rapa spp. pekinensis) and cabbage (Brassica oleracea var. capitata). Both Cas9-NG and SpG induced efficient mutagenesis at NGN PAMs, while SpG outperformed Cas9-NG at NGC and NGT PAMs. SpRY achieved efficient editing at almost all PAMs (NRN > NYN), albeit with some self-targeting activity at transfer (T)-DNA sequences. And SpRY-induced mutants were detected in cabbage plants in a PAM-less fashion. Moreover, an adenine base editor was developed using SpRY and TadA8e deaminase that induced A-to-G conversions within target sites using non-canonical PAMs. Together, the toolboxes developed here induced successful genome editing in Chinese cabbage and cabbage. Our work further expands the targeting scope of genome editing and paves the way for future basic research and genetic improvement in Brassica. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00155-7.

High-Throughput Screening of PAM-Flexible Cas9 Variants for Expanded Genome Editing in the Silkworm (Bombyx mori).

Genome editing provides novel opportunities for the precise genome engineering of diverse organisms. Significant progress has been made in the development of genome-editing tools for Bombyx mori (B. mori) in recent years. Among these, CRISPR/Cas9, which is currently the most commonly used system in lepidopteran insects, recognizes NGG protospacer adjacent motif (PAM) sequences within the target locus. However, Cas9 lacks the ability to target all gene loci in B. mori, indicating the need for Cas9 variants with a larger editing range. In this study, we developed a high-throughput screening platform to validate Cas9 variants at all possible recognizable and editable PAM sites for target sequences in B. mori. This platform enabled us to identify PAM sites that can be recognized by both xCas9 3.7 and SpCas9-NG variants in B. mori and to assess their editing efficiency. Cas9 shows PAM sites every 13 base pairs in the genome, whereas xCas9 3.7 and SpCas9-NG have an average distance of 3.4 and 3.6 base pairs, respectively, between two specific targeting sites. Combining the two Cas9 variants could significantly expand the targeting range of the genome, accelerate research on the B. mori genome, and extend the high-throughput rapid screening platform to other insects, particularly those lacking suitable NGG PAM sequences.

Engineering Cas9: next generation of genomic editors.

The Cas9 endonuclease of the CRISPR/Cas type IIA system from Streptococcus pyogenes is the heart of genome editing technology that can be used to treat human genetic and viral diseases. Despite its large size and other drawbacks, S. pyogenes Cas9 remains the most widely used genome editor. A vast amount of research is aimed at improving Cas9 as a promising genetic therapy. Strategies include directed evolution of the Cas9 protein, rational design, and domain swapping. The first generation of Cas9 editors comes directly from the wild-type protein. The next generation is obtained by combining mutations from the first-generation variants, adding new mutations to them, or refining mutations. This review summarizes and discusses recent advances and ways in the creation of next-generation genomic editors derived from S. pyogenes Cas9. KEY POINTS: • The next-generation Cas9-based editors are more active than in the first one. • PAM-relaxed variants of Cas9 are improved by increased specificity and activity. • Less mutagenic and immunogenic variants of Cas9 are created.

Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing.

The increasing emergence of Cas9 variants has attracted broad interest, as these variants were designed to expand CRISPR applications. New Cas9 variants typically feature higher editing efficiency, improved editing specificity, or alternative PAM sequences. To select Cas9 variants and gRNAs for high-fidelity and efficient genome editing, it is crucial to systematically quantify the editing performances of gRNAs and develop prediction models based on high-quality datasets. Using synthetic gRNA-target paired libraries and next-generation sequencing, we compared the activity and specificity of gRNAs of four SpCas9 variants. The nucleotide composition in the PAM-distal region had more influence on the editing efficiency of HiFi Cas9 and LZ3 Cas9. We further developed machine learning models to predict the gRNA efficiency and specificity for the four Cas9 variants. To aid users from broad research areas, the machine learning models for the predictions of gRNA editing efficiency within human genome sites are available on our website.

Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management.

The innovative advances in transforming clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) into different variants have taken the art of genome-editing specificity to new heights. Allosteric modulation of Cas9-targeting specificity by sgRNA sequence alterations and protospacer adjacent motif (PAM) modifications have been a good lesson to learn about specificity and activity scores in different Cas9 variants. Some of the high-fidelity Cas9 variants have been ranked as Sniper-Cas9, eSpCas9 (1.1), SpCas9-HF1, HypaCas9, xCas9, and evoCas9. However, the selection of an ideal Cas9 variant for a given target sequence remains a challenging task. A safe and efficient delivery system for the CRISPR/Cas9 complex at tumor target sites faces considerable challenges, and nanotechnology-based stimuli-responsive delivery approaches have significantly contributed to cancer management. Recent innovations in nanoformulation design, such as pH, glutathione (GSH), photo, thermal, and magnetic responsive systems, have modernized the art of CRISPR/Cas9 delivery approaches. These nanoformulations possess enhanced cellular internalization, endosomal membrane disruption/bypass, and controlled release. In this review, we aim to elaborate on different CRISPR/Cas9 variants and advances in stimuli-responsive nanoformulations for the specific delivery of this endonuclease system. Furthermore, the critical constraints of this endonuclease system on clinical translations towards the management of cancer and prospects are described.

Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.

Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.

Expanding PAM recognition and enhancing base editing activity of Cas9 variants with non-PI domain mutations derived from xCas9.

The recognition of protospacer adjacent motif (PAM) is a key factor for the CRISPR (i.e. clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (Cas9) system to distinguish foreign DNAs from the host genome, and also significantly restricts the targeting scope of the system during genome-editing applications. Structurally, the PAM interacting (PI) domain, which usually is located in the C-terminus of Cas9 proteins, directly binds to PAM and plays a key role in determining the recognition specificity. However, several lines of evidence showed that other regions of Cas9 protein beyond the PI domain might also play roles in PAM interaction. Here, we constructed a mosaic SpCas9 protein (xCas9-NG) by fusing the PI domain of SpCas9 PAM variant, Cas9-NG with the non-PI fragment of xCas9 protein that contains multiple amino acid substitutions. We found that non-PI fragment of xCas9 expanded PAM recognition of the Cas9-NG PI domain. In addition, xCas9-NG showed an improved editing efficiency in the majority of targets harboring xCas9 and Cas9-NG PAMs. Importantly, this finding was also successfully extended to other Cas9 variants, including SpRY and the non-G SpCas9 series. Together, our work expands the target scope of SpCas9 editing system and demonstrates the notion that the non-PI domain fragment plays an important role in PAM restriction.

PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.

Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing.

The repurposing of the CRISPR/Cas bacterial defense system against bacteriophages as simple and flexible molecular tools has revolutionized the field of gene editing. These tools are now widely used in basic research and clinical trials involving human somatic cells. However, a global moratorium on all clinical uses of human germline editing has been proposed because the technology still lacks the required efficacy and safety. Here we focus on the approaches developed since 2013 to decrease the frequency of unwanted mutations (the off-targets) during CRISPR-based gene editing.

In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems.

CRISPR/Cas9 has revolutionized the ability to edit cellular DNA and is poised to transform the treatment of genetic diseases. One of the major concerns regarding its therapeutic use is the potential for off-target DNA cleavage, which could have detrimental consequences in vivo. To circumvent this, a number of strategies have been employed to develop next-generation CRISPR/Cas9 systems with improved specificity. These include the development of new protein variants of Cas9, as well as chemically modified guide RNA molecules. Here, we provide detailed protocols for two in vitro methods that enable the specificity of first- and next-generation CRISPR/Cas9 systems to be compared, and we demonstrate their applicability to evaluating chemically modified guide RNAs. One of these assays allows the specificity of different guide RNA/Cas9 complexes to be compared on a set of known off-target DNA sequences, while the second provides a broad specificity profile based on cleavage of a massive library of potential off-target DNA sequences. Collectively, these assays may be used to evaluate the specificity of different CRISPR/Cas9 systems on any DNA target sequence in a time- and cost-effective manner.

Simplified adenine base editors improve adenine base editing efficiency in rice.

Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE-P1S (Adenine Base Editor-Plant version 1 Simplified) containing the ecTadA*7.10-nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE-P1 consisting of the ecTadA-ecTadA*7.10-nSpCas9 (D10A) fusion. We found that the protein expression level of ABE-P1S is higher than that of ABE-P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE-P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10-nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH-Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.

Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.

BACKGROUND: Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. RESULTS: We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. CONCLUSIONS: We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

The potential application of genome editing by using CRISPR/Cas9, and its engineered and ortholog variants for studying the transcription factors involved in the maintenance of phosphate homeostasis in model plants.

Phosphorus (P), an essential macronutrient, is pivotal for growth and development of plants. Availability of phosphate (Pi), the only assimilable P, is often suboptimal in rhizospheres. Pi deficiency triggers an array of spatiotemporal adaptive responses including the differential regulation of several transcription factors (TFs). Studies on MYB TF PHR1 in Arabidopsis thaliana (Arabidopsis) and its orthologs OsPHRs in Oryza sativa (rice) have provided empirical evidence of their significant roles in the maintenance of Pi homeostasis. Since the functional characterization of PHR1 in 2001, several other TFs have now been identified in these model plants. This raised a pertinent question whether there are any likely interactions across these TFs. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has provided an attractive paradigm for editing genome in plants. Here, we review the applications and challenges of this technique for genome editing of the TFs for deciphering the function and plausible interactions across them. This technology could thus provide a much-needed fillip towards engineering TFs for generating Pi use efficient plants for sustainable agriculture. Furthermore, we contemplate whether this technology could be a viable alternative to the controversial genetically modified (GM) rice or it may also eventually embroil into a limbo.

Expanding the base editing scope in rice by using Cas9 variants.

Base editing is a novel genome editing strategy that enables irreversible base conversion at target loci without the need for double stranded break induction or homology-directed repair. Here, we developed new adenine and cytosine base editors with engineered SpCas9 and SaCas9 variants that substantially expand the targetable sites in the rice genome. These new base editors can edit endogenous genes in the rice genome with various efficiencies. Moreover, we show that adenine and cytosine base editing can be simultaneously executed in rice. The new base editors described here will be useful in rice functional genomics research and will advance precision molecular breeding in crops.

Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

Rescue of high-specificity Cas9 variants using sgRNAs with matched 5' nucleotides.

We report that engineered Cas9 variants with improved specificity-eCas9-1.1 and Cas9-HF1-are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5' terminus, relative to target DNA sequences. Because the nucleotide at the 5' end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5' nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.