ABSTRACT
CRISPR-Cas12a systems are becoming an attractive genome editing tool for cell engineering due to their broader editing capabilities compared to CRISPR-Cas9 counterparts. As opposed to Cas9, the Cas12a endonucleases are characterized by a lack of trans-activating crRNA (tracrRNA), which reduces the complexity of the editing system and simultaneously makes CRISPR RNA (crRNA) engineering a promising approach toward further improving and modulating editing activity of the CRISPR-Cas12a systems. Here, we design and validate sixteen types of structurally engineered Cas12a crRNAs targeting various immunologically relevant loci in-vitro and in-cellulo. We show that all our structural modifications in the loop region, ranging from engineered breaks (STAR-crRNAs) to large gaps (Gap-crRNAs), as well as nucleotide substitutions, enable gene-cutting in the presence of various Cas12a nucleases. Moreover, we observe similar insertion rates of short HDR templates using the engineered crRNAs compared to the wild-type crRNAs, further demonstrating that the introduced modifications in the loop region led to comparable genome editing efficiencies. In conclusion, we show that Cas12a nucleases can broadly utilize structurally engineered crRNAs with breaks or gaps in the otherwise highly-conserved loop region, which could further facilitate a wide range of genome editing applications.
Subject(s)
CRISPR-Cas Systems , RNA, Guide, Kinetoplastida , Endonucleases/genetics , Endonucleases/metabolism , Gene Editing , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolismABSTRACT
A DBR tapered diode laser in continuous wave operation was used to generate second-harmonic radiation at 589 nm in a PPMgO:LN ridge waveguide crystal. An optical output power of 0.86 W at an optical-to-optical and an electrical-to-optical efficiency of 42% and 11%, respectively, was achieved. The visible radiation was characterized by a spectral bandwidth ΔνFWHM of 230 MHz and a beam propagation parameter M1/e22 better than 1.1. The integration of such a system into a housing of a small footprint will enable a portable and highly efficient module featuring a visible output power in the watt-level range.
ABSTRACT
In this work, frequency doubling of a passively mode-locked 3.5 mm long monolithic distributed Bragg reflector diode laser is investigated experimentally. At 1064 nm, optical pulses with a duration of 12.4 ps are generated at a repetition rate of 13 GHz and a peak power of 825 mW, resulting in an average power of 133 mW. Second-harmonic generation is carried out in a periodically poled MgO-doped LiNbO3 ridge waveguide at a normalized nonlinear conversion efficiency of 930%/W. A maximum average second-harmonic power of 40.9 mW, corresponding to a pulse energy of 3.15 pJ, is reached in the experiment at an opto-optical conversion efficiency of 30.8%. The normalized nonlinear conversion efficiency in mode-locked operation is more than 2 times larger compared to continuous-wave operation.
