Engineering of CRISPR-Cas12b for Human Genome Editing

Overview

Journal Nat Commun

Specialty Biology

Date 2019 Jan 24

PMID 30670702

Citations 147

Authors

Jonathan Strecker

Sara Jones

Balwina Koopal

Jonathan Schmid-Burgk

Bernd Zetsche

Linyi Gao

Kira S Makarova

Eugene V Koonin

Feng Zhang

Affiliations

Soon will be listed here.

Abstract

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

Citing Articles

CRISPR-Cas applications in agriculture and plant research.

Tuncel A, Pan C, Clem J, Liu D, Qi Y Nat Rev Mol Cell Biol. 2025; .

PMID: 40055491 DOI: 10.1038/s41580-025-00834-3.

Exploiting the Specificity of CRISPR/Cas System for Nucleic Acids Amplification-Free Disease Diagnostics in the Point-of-Care.

Yee B, Ali N, Mohd-Naim N, Ahmed M Chem Bio Eng. 2025; 1(4):330-339.

PMID: 39974464 PMC: 11835143. DOI: 10.1021/cbe.3c00112.

Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM.

Zheng W, Li H, Liu M, Wei Y, Liu B, Li Z Signal Transduct Target Ther. 2025; 10(1):66.

PMID: 39955288 PMC: 11830025. DOI: 10.1038/s41392-025-02147-5.

Nano-Polymers as Cas9 Inhibitors.

Chepurna O, Chatterjee A, Li Y, Ding H, Murali R, Black K Polymers (Basel). 2025; 17(3).

PMID: 39940619 PMC: 11820846. DOI: 10.3390/polym17030417.

Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data.

Nayfach S, Bhatnagar A, Novichkov A, Estevam G, Kim N, Hill E bioRxiv. 2025; .

PMID: 39829748 PMC: 11741284. DOI: 10.1101/2025.01.06.631536.

References

Wu D, Guan X, Zhu Y, Ren K, Huang Z . Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA. Cell Res. 2017; 27(5):705-708. PMC: 5520857. DOI: 10.1038/cr.2017.46. View

Yang H, Gao P, Rajashankar K, Patel D . PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease. Cell. 2016; 167(7):1814-1828.e12. PMC: 5278635. DOI: 10.1016/j.cell.2016.11.053. View

Schmid-Burgk J, Schmidt T, Gaidt M, Pelka K, Latz E, Ebert T . OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines. Genome Res. 2014; 24(10):1719-23. PMC: 4199374. DOI: 10.1101/gr.176701.114. View

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View

Hsu P, Scott D, Weinstein J, Ran F, Konermann S, Agarwala V . DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013; 31(9):827-32. PMC: 3969858. DOI: 10.1038/nbt.2647. View

Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W . Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol. 2017; 15(3):169-182. PMC: 5851899. DOI: 10.1038/nrmicro.2016.184. View

Liu L, Chen P, Wang M, Li X, Wang J, Yin M . C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism. Mol Cell. 2016; 65(2):310-322. DOI: 10.1016/j.molcel.2016.11.040. View

Makarova K, Grishin N, Shabalina S, Wolf Y, Koonin E . A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006; 1:7. PMC: 1462988. DOI: 10.1186/1745-6150-1-7. View

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

10.

Fu Y, Foden J, Khayter C, Maeder M, Reyon D, Joung J . High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31(9):822-6. PMC: 3773023. DOI: 10.1038/nbt.2623. View

11.

Shmakov S, Abudayyeh O, Makarova K, Wolf Y, Gootenberg J, Semenova E . Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell. 2015; 60(3):385-97. PMC: 4660269. DOI: 10.1016/j.molcel.2015.10.008. View

12.

Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q . Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov. 2018; 4:63. PMC: 6255809. DOI: 10.1038/s41421-018-0069-3. View

13.

Tsai S, Zheng Z, Nguyen N, Liebers M, Topkar V, Thapar V . GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2014; 33(2):187-197. PMC: 4320685. DOI: 10.1038/nbt.3117. View

14.

Suzuki M . A framework for the DNA-protein recognition code of the probe helix in transcription factors: the chemical and stereochemical rules. Structure. 1994; 2(4):317-26. DOI: 10.1016/s0969-2126(00)00033-2. View

15.

Leenay R, Maksimchuk K, Slotkowski R, Agrawal R, Gomaa A, Briner A . Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. Mol Cell. 2016; 62(1):137-47. PMC: 4826307. DOI: 10.1016/j.molcel.2016.02.031. View

16.

Knott G, Doudna J . CRISPR-Cas guides the future of genetic engineering. Science. 2018; 361(6405):866-869. PMC: 6455913. DOI: 10.1126/science.aat5011. View

17.

Saavedra H, Wrabl J, Anderson J, Li J, Hilser V . Dynamic allostery can drive cold adaptation in enzymes. Nature. 2018; 558(7709):324-328. PMC: 6033628. DOI: 10.1038/s41586-018-0183-2. View

18.

Seuylemezian A, Cooper K, Schubert W, Vaishampayan P . Draft Genome Sequences of 12 Dry-Heat-Resistant Bacillus Strains Isolated from the Cleanrooms Where the Viking Spacecraft Were Assembled. Genome Announc. 2018; 6(12). PMC: 5864948. DOI: 10.1128/genomeA.00094-18. View

19.

Schmid-Burgk J, Hornung V . BrowserGenome.org: web-based RNA-seq data analysis and visualization. Nat Methods. 2015; 12(11):1001. DOI: 10.1038/nmeth.3615. View

20.

Mavromatis K, Tsigos I, Tzanodaskalaki M, Kokkinidis M, Bouriotis V . Exploring the role of a glycine cluster in cold adaptation of an alkaline phosphatase. Eur J Biochem. 2002; 269(9):2330-5. DOI: 10.1046/j.1432-1033.2002.02895.x. View