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Updated: Mar 1, 2026

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

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Published on: April 26, 2017

Engineered Cpf1 variants with altered PAM specificities.

Linyi Gao^1,2, David B T Cox^1,3,4, Winston X Yan^1,4,5

¹Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

Nature Biotechnology

|June 6, 2017

Summary

This summary is machine-generated.

We engineered novel variants of the RNA-guided endonuclease Cpf1 (CRISPR-Cas12a) to expand its genome editing capabilities. These modified Cpf1 enzymes recognize new PAM sequences, significantly increasing targeting range in human cells.

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Published on: February 17, 2023

Area of Science:

Molecular Biology
Genome Engineering
Biotechnology

Background:

Cpf1 (CRISPR-Cas12a) is a versatile RNA-guided endonuclease for genome editing.
Current Cpf1 systems, like AsCpf1 and LbCpf1, are limited by their TTTV protospacer adjacent motif (PAM) requirement.
Expanding Cpf1's PAM compatibility is crucial for broader genome editing applications.

Purpose of the Study:

To engineer Cpf1 variants with altered PAM specificities to broaden targeting range.
To enhance the utility of Cpf1 for genome editing in eukaryotic systems, particularly human cells.

Main Methods:

Structure-guided mutagenesis screen of Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1).
In vitro and in human cell assays to evaluate variant activity and specificity.
Genome-wide off-target analysis using BLISS (Beyond Localized Integration of Sequence and Structure).

Main Results:

Two AsCpf1 variants recognizing TYCV and TATV PAMs were engineered with enhanced activity.
These variants demonstrated high DNA-targeting specificity, with further improvement via an additional mutation.
Similar PAM specificity alterations were achieved in Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1).

Conclusions:

Engineered Cpf1 variants significantly expand the targeting range of CRISPR-Cas12a systems.
The expanded targeting range increases Cpf1 utility by approximately threefold in human coding sequences.
These advancements offer greater flexibility for precise genome editing applications.