Comparative optimization of combinatorial CRISPR screens

Li, Ruitong; Klingbeil, Olaf; Monducci, Davide; Young, M. J. L.; Rodriguez, Diego J.; Bayyat, Zaid; Dempster, Joshua M.; Kesar, Devishi; Yang, Xiaoping; Zamanighomi, Mahdi; Vakoc, Christopher R.; Ito, Takahiro; Sellers, William R.

doi:10.1038/s41467-022-30196-9

Cited by 25 publications

(19 citation statements)

References 39 publications

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“…We compared combinatorial SpCas9, AsCas12a, and orthogonal CHyMErA screening approaches by targeting the same gene set under comparable experimental conditions and observed that single and paired gRNA effect size ranges are the largest with SpCas9. In line with our findings, the gRNA effect size range of AsCas12a was reported to be low 29 , and even though AsCas12a has been engineered and optimized to perform in CRISPR screens, also the recent literature indicates that AsCas12a is not as robustly performing as SpCas9 20 , 32 . However, the low effect size range associated with enAsCas12a and CHyMErA may be attributed to either difference in nuclease activity or the lack of respective highly-active spacer sequences.…”

Section: Discussionsupporting

confidence: 90%

“…While combinatorial CRISPR screens are powerful, they suffer from scalability issues related to large numbers of to-be-investigated query genes 20 . In line with that, we have previously demonstrated that, besides library diversity, library uniformity is a critical size-determining factor of the experimental scale, with uniform libraries (distribution skew; the ratio between the 10th and 90th percentile of normalized read counts is below 2.5) supporting experimental down-scaling and improving scalability and feasibility 13 , 21 .…”

Section: Introductionmentioning

confidence: 99%

“…uniformity, completeness), different Cas nucleases and gRNA-expression systems are available for combinatorial screens, with none of them or combinations thereof, yet being robustly established for combinatorial or orthogonal approaches. This is, in part, attributed to the overall lack of quantitative data comparing combinatorial CRISPR technologies 9 , 19 , 20 .…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Optimized metrics for orthogonal combinatorial CRISPR screens

Cetin,

Wegner,

Luwisch

et al. 2023

Sci Rep

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CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Optimized metrics for orthogonal combinatorial CRISPR screens

Cetin,

Wegner,

Luwisch

et al. 2023

Sci Rep

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“…As the rate of swapping is distance-dependent, this concern is minimized with the Cas12a architecture as individual guides are separated by only a 20 nucleotide direct repeat (DR), compared to several hundred nucleotides with Cas9 guide cassettes. We and others have leveraged these advantages of Cas12a to explore synthetic lethality 19,24 and paralog redundancy 25,26 by targeting multiple genes simultaneously.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

Griffith

Zheng

McGee

et al. 2023

Preprint

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Cas12a CRISPR technology, unlike Cas9, allows for multiplexing guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and we provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

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“…Another approach to reducing CRISPR library size is by multiplexing two or more guides per gene on a single construct, which enables functional redundancy to compensate for suboptimal gene knockout by individual guides. Multiplexing CRISPR/Cas9 guides on a single construct is challenging in that it requires multistep cloning for library construction (16) and is prone to guide uncoupling through recombination ( 17, 18 ). In contrast, the alternative Cas12a (Cpf1) is distinct in its ability to specifically and efficiently process multiplexed guide arrays for genome editing ( 19–21 ).…”

Section: Introductionmentioning

confidence: 99%

Optimization of genomewide CRISPR screens using AsCas12a and multi-guide arrays

Petiwala

Modi

Anton

et al. 2022

Preprint

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Genomewide loss-of-function (LOF) screening using CRISPR (clustered regularly interspaced short palindromic repeats) has facilitated the discovery of novel gene functions across diverse physiological and pathophysiological systems. A challenge with conventional genomewide CRISPR/Cas9 libraries is the unwieldy size (60,000 to 120,000 constructs), which is resource intensive and prohibitive in some experimental contexts. One solution to streamlining CRISPR screening is by multiplexing two or more guides per gene on a single construct, which enables functional redundancy to compensate for suboptimal gene knockout by individual guides. In this regard, AsCas12a (Cpf1) and its derivatives, e.g., enhanced AsCas12a (enAsCas12a), have enabled multiplexed guide arrays to be specifically and efficiently processed for genome editing. Prior studies have established that multiplexed CRISPR/Cas12a libraries perform comparably to the larger equivalent CRISPR/Cas9 libraries, yet the most efficient CRISPR/Cas12a library design remains unresolved. Here, we demonstrate that CRISPR/Cas12a genomewide LOF screening performed optimally with three guides arrayed per gene construct and could be adapted to robotic cell culture without noticeable differences in screen performance. Thus, the conclusions from this study provide novel insight to streamlining genomewide LOF screening using CRISPR/Cas12a and robotic cell culture.

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Comparative optimization of combinatorial CRISPR screens

Cited by 25 publications

References 39 publications

Optimized metrics for orthogonal combinatorial CRISPR screens

Optimized metrics for orthogonal combinatorial CRISPR screens

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

Optimization of genomewide CRISPR screens using AsCas12a and multi-guide arrays

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