Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning

Koblan, Luke W.; Arbab, Mandana; Shen, Max W.; Hussmann, Jeffrey A.; Anzalone, Andrew V.; Doman, Jordan L.; Newby, Gregory A.; Yang, Dian; Mok, Beverly; Replogle, Joseph M.; Xu, Albert; Sisley, Tyler A.; Weissman, Jonathan S.; Adamson, Britt; Liu, David R.

doi:10.1038/s41587-021-00938-z

Cited by 146 publications

(87 citation statements)

References 43 publications

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“…It might be possible to resolve the mechanism through mining a large editing data set. While we were preparing this manuscript, a recent study reported a similar phenomenon in human cells (Koblan et al, 2021). The authors only observed moderately improved C-to-G editing efficiency after replacing the E. coli UNG with a UNG ortholog from Mycobacterium smegmatis (UdgX).…”

Section: Discussionmentioning

confidence: 67%

“…Interestingly, no single CGBE outperformed other CGBEs at all target sites, echoing our findings in plants. The authors ended up using machine learning to develop a program termed CGBE-Hive for predicting the performance of individual CGBEs based on a large amount of editing data generated in human cells ( Koblan et al, 2021 ). Thus, it is envisioned that a similar approach in plants may be needed for understanding the editing preference of CGBEs in plants to advance the use of C-to-G editing and improve reliability to aid basic and applied plant research.…”

Section: Discussionmentioning

confidence: 99%

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Exploring C-To-G Base Editing in Rice, Tomato, and Poplar

Sretenovic

Liu

et al. 2021

Front. Genome Ed.

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As a precise genome editing technology, base editing is broadly used in both basic and applied plant research. Cytosine base editors (CBEs) and adenine base editors (ABEs) represent the two commonly used base editor types that mediate C-to-T and A-to-G base transition changes at the target sites, respectively. To date, no transversion base editors have been described in plants. Here, we assessed three C-to-G base editors (CGBEs) for targeting sequences with SpCas9’s canonical NGG protospacer adjacent motifs (PAMs) as well as three PAM-less SpRY-based CGBEs for targeting sequences with relaxed PAM requirements. The analyses in rice and tomato protoplasts showed that these CGBEs could make C-to-G conversions at the target sites, and they preferentially edited the C6 position in the 20-nucleotide target sequence. C-to-T edits, insertions and deletions (indels) were major byproducts induced by these CGBEs in the protoplast systems. Further assessment of these CGBEs in stably transformed rice and poplar plants revealed the preference for editing of non-GC sites, and C-to-T edits are major byproducts. Successful C-to-G editing in stably transgenic rice plants was achieved by rXRCC1-based CGBEs with monoallelic editing efficiencies up to 38% in T0 lines. The UNG-rAPOBEC1 (R33A)-based CGBE resulted in successful C-to-G editing in polar, with monoallelic editing efficiencies up to 6.25% in T0 lines. Overall, this study revealed that different CGBEs have different preference on preferred editing sequence context, which could be influenced by cell cycles, DNA repair pathways, and plant species.

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Exploring C-To-G Base Editing in Rice, Tomato, and Poplar

Sretenovic

Liu

et al. 2021

Front. Genome Ed.

View full text Add to dashboard Cite

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“…Sequence identity was important for several unintended substitutions as well (Figure S1I). In particular, C to G transversions by cytosine editors increased over 4-fold at the TCT motif, and were more frequent when the C was followed by a T. This effect was recently used to develop C to G editors elsewhere [32][33][34]. Furthermore, there is evidence that the cytosine base editors also operate on the opposite strand, as G to A edits were found in much greater quantity in cytosine editors than in controls with only wild-type Cas9 (Figure 1C), and these edits also mirrored motif preferences, albeit with lesser effect on the opposite strand (300% increase in TC to TT in BE4, 50% increase in GA to AA).…”

Section: Resultsmentioning

confidence: 99%

Predicting base editing outcomes using position-specific sequence determinants

Pallaseni

Peets

Koeppel

et al. 2021

Preprint

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Nucleotide-level control over DNA sequences is poised to power functional genomics studies and lead to new therapeutics. CRISPR/Cas base editors promise to achieve this ability, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ~14,000 target sequences. Base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base, and is correlated with measures of Cas9 guide RNA efficiency. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, with a preceding thymine in both editor types leading to a wider editing window, while a preceding guanine in cytosine editors and preceding adenine in adenine editors to a narrower one. The impact of features on editing rate depends on the position, with guide RNA efficacy mainly influencing bases around the centre of the window, and sequence biases away from it. We use these observations to train a machine learning model to predict editing activity per position for both adenine and cytosine editors, with accuracy ranging from 0.49 to 0.72 between editors, and with better generalization performance across datasets than existing tools. We demonstrate the usefulness of our model by predicting the efficacy of potential disease mutation correcting guides, and find that most of them suffer from more unwanted editing than corrected outcomes. This work unravels the position-specificity of base editing biases, and provides a solution to account for them, thus allowing more efficient planning of base edits in experimental and therapeutic contexts.

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“…Indeed, due to the relatively high prevalence of false-negatives with the technology, especially in negative-selection screens, the benefit of added depth is worthwhile, enabling the use of multiple unique guides to pinpoint regions of particular interest in a target locus. The recent development of a nearly-PAM-less Cas9 variant 26 , as well as approaches to generate C>G edits 52–55 , suggests that more editing outcomes and thus even finer resolution will be possible for base editor screens. Generating a library of every possible amino acid substitution for an entire open reading frame is certainly possible 56–58 , but is also expensive.…”

Section: Discussionmentioning

confidence: 99%

Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

Sangree

Griffith

Szegletesh

et al. 2021

Preprint

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Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we benchmark PAM preferences, on-target activity, and off-target susceptibility of 11 variants of SpCas9 in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A>G and C>T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new insights into these clinically-relevant genes. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.

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Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning

Cited by 146 publications

References 43 publications

Exploring C-To-G Base Editing in Rice, Tomato, and Poplar

Exploring C-To-G Base Editing in Rice, Tomato, and Poplar

Predicting base editing outcomes using position-specific sequence determinants

Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

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