Genome-wide specificity profiles of CRISPR-Cas Cpf1 nucleases in human cells
Bibliographic record
Abstract
CRISPR-Cas Cpf1 nucleases have recently been described as an alternative genomeediting platform 1 , yet their activities and genome-wide specificities remain largely undefined.Here we show that two Cpf1 nucleases function robustly in human cells with on-target efficiencies comparable to those of the widely used Streptococcus pyogenes Cas9 (SpCas9) [2][3][4][5] .We also demonstrate that four to six bases at the 3' end of the short CRISPR RNA (crRNA) used to program Cpf1 are insensitive to single base mismatches but that many of the other bases within the crRNA targeting region are highly sensitive to single or double substitutions.Consistent with these results, GUIDE-seq and targeted deep sequencing analyses of two Cpf1 nucleases revealed no detectable off-target cleavage for over half of 20 different crRNAs we examined.Our results suggest that the two Cpf1 nucleases we characterized generally possess high specificities in human cells, a finding that should encourage broader use of these genome editing enzymes.Clustered, regularly interspaced, short palindromic repeat (CRISPR) systems encode RNA-guided endonucleases that are essential for bacterial adaptive immunity 6 .Much work has shown that these CRISPR-associated (Cas) nucleases can be easily programmed to cleave target DNA sequences of interest for genome editing applications in a variety of different organisms 2-5 .One class of these nucleases, known as Cas9 proteins, naturally complex with two short RNAs: a crRNA and a trans-activating crRNA (tracrRNA) 7,8 .SpCas9, the most commonly used Cas9 orthologue, uses a crRNA harboring 20 nucleotides at its 5' end with complementarity to the "protospacer" region of its target DNA site.Efficient cleavage also requires the presence of a protospacer adjacent motif (PAM) which is recognized by SpCas9.The crRNA and tracrRNA are now typically combined into a single ~100 nt guide RNA (gRNA) 7, 9-11 , which functions efficiently with SpCas9 to direct its cleavage activity.The genome-wide specificities of SpCas9 nucleases paired with various gRNAs have been well-characterized .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".