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CRISPR-Cas9-AID base editor is a powerful gain-of-function screening tool.
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Predictable and precise template-free CRISPR editing of pathogenic variants.
Following Cas9 cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9 editing is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. We constructed a library of 2,000 Cas9 guide RNAs paired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines. inDelphi predicts that 5-11% of Cas9 guide RNAs targeting the human genome are 'precise-50', yielding a single genotype comprising greater than or equal to 50% of all major editing products. We experimentally confirmed precise-50 insertions and deletions in 195 human disease-relevant alleles, including correction in primary patient-derived fibroblasts of pathogenic alleles to wild-type genotype for Hermansky-Pudlak syndrome and Menkes disease. This study establishes an approach for precise, template-free genome editing.
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PhieDBEs: a DBD-containing, PAM-flexible, high-efficiency dual base editor toolbox with wide targeting scope for use in plants.
Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.
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Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes.
Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.
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CRISPR/Cas9 can mediate high-efficiency off-target mutations in mice in vivo.
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Screening an Effective Dual-Adeno-Associated Virus Split-Cytosine Base Editor System for C-to-T Conversion In Vivo.
The cytosine base editor (CBE) has shown promise as a gene editing tool for gene therapy, as it can convert cytidine to thymidine. Adeno-associated virus (AAV) has been widely used for in vivo gene therapy, but its limited 4.7 kb packing capacity presents challenges in delivering CBE by a single AAV. To address this, one feasible solution is to split CBE into two sections for dual-AAV delivery. In this study, we utilized BE3 as an example and constructed 22 potential split-BE3 pairs with the combination of 11 splitting sites and two split-inteins (Npu and Rma). These split-BE3 pairs were initially screened in the green fluorescent protein (GFP) reporter system, with six split-BE3 pairs selected for further evaluation. The subsequent screening of split-BE3 pairs was performed at two endogenous sites in 293T and HeLa cells, revealing that the split-BE3-Rma674, split-BE3-Rma713, and split-BE3-Rma1005 displayed effective C-to-T conversion after transfection. The effectiveness of dual-AAV split-BE3 was further validated in culture cells and adult mouse eyes. Of note, the split-BE3-Rma674 demonstrated the most efficient C-to-T editing after AAV infection, with a maximal editing efficiency of 23.29% ± 10.98% in the mouse retinal pigment epithelium cells in vivo. Overall, our study presents a novel split-BE3 system with effective C-to-T conversion, which could be applied to CBE-based in vivo gene therapy.
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New Editing Tools for Gene Therapy in Inherited Retinal Dystrophies.
Inherited retinal dystrophies (IRDs) are a heterogeneous group of diseases that affect more than 2 million people worldwide. Gene therapy (GT) has emerged as an exciting treatment modality with the potential to provide long-term benefit to patients. Today, gene addition is the most straightforward GT for autosomal recessive IRDs. However, there are three scenarios where this approach falls short. First, in autosomal dominant diseases caused by gain-of-function or dominant-negative mutations, the toxic mutated protein needs to be silenced. Second, a number of IRD genes exceed the limited carrying capacity of adeno-associated virus vectors. Third, there are still about 30% of patients with unknown mutations. In the first two contexts, precise editing tools, such as CRISPR-Cas9, base editors, or prime editors, are emerging as potential GT solutions for the treatment of IRDs. Here, we review gene editing tools based on CRISPR-Cas9 technology that have been used in vivo and the recent first-in-human application of CRISPR-Cas9 in an IRD.
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A high-efficient and naked-eye visible CRISPR/Cas9 system in Arabidopsis.
Introducing 35S-dsRED2 into the Cas9 vector which expresses naked-eye visible dsRED2 greatly facilitates the genetic screening, and the WUS promoter driving the Cas9 expression can improve editing efficiency in Arabidopsis. CRISPR/Cas9-dependent genome editing has been applied to generate random insertions and deletions, targeted insertions or replacements, and precise base changes for both fundamental studies in many plant species and crop improvement. To simplify the screening procedure for target gene-edited transformants, we introduced a CaMV 35S-driven dsRED2 cassette (35S-dsRED2) into the Cas9 vector to express the naked-eye visible protein dsRED2, which can be observed under white light, greatly facilitated the genetic screening and reduced labor intensity without using any instrument. In addition, the WUS promoter was used to drive the expression of Cas9, which successfully improved the target genes editing efficiency and enabled the homozygous mutagenesis of two genes in T1 generation in Arabidopsis. Considering the conserved function and expression pattern of WUS across the plant species, this dsRED2-WUS/Cas9 system could also be used in many crops.
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Generation of genetically modified mice using SpCas9-NG engineered nuclease.
Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5'-NGG sequence, SpCas9-NG recognized the 5'-NGA, 5'-NGC and 5'-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.
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CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.
Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.
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